Assays for detecting neutralizing autoantibodies to biologic therapy

ABSTRACT

The present invention provides assays for detecting and measuring the presence or level of neutralizing and non-neutralizing autoantibodies to biologics such as anti-TNFα drug therapeutics in a sample. The present invention is useful for monitoring the formation of neutralizing and/or non-neutralizing anti-drug antibodies over time while a subject is on biologic therapy. The present invention is also useful for predicting and/or determining the cross-reactivity of neutralizing anti-drug antibodies in a subject&#39;s sample with alternative biologic therapies. As such, the present invention provides information for guiding treatment decisions for those subjects receiving therapy with a biologic agent and improves the accuracy of optimizing therapy, reducing toxicity, and/or monitoring the efficacy of therapeutic treatment to biologic therapy.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of PCT/US2012/045794, filed Jul. 6,2012, which application claims priority to U.S. Provisional ApplicationNo. 61/505,031, filed Jul. 6, 2011, U.S. Provisional Application No.61/528,072, filed Aug. 26, 2011, and U.S. Provisional Application No.61/535,884, filed Sep. 16, 2011, the disclosures of which are herebyincorporated by reference in their entirety for all purposes.

BACKGROUND OF THE INVENTION

Autoimmune disorders are a significant and widespread medical problem.For example, rheumatoid arthritis (RA) is an autoimmune diseaseaffecting more than two million people in the United States. RA causeschronic inflammation of the joints and typically is a progressiveillness that has the potential to cause joint destruction and functionaldisability. The cause of rheumatoid arthritis is unknown, althoughgenetic predisposition, infectious agents and environmental factors haveall been implicated in the etiology of the disease. In active RA,symptoms can include fatigue, lack of appetite, low grade fever, muscleand joint aches and stiffness. Also during disease flare ups, jointsfrequently become red, swollen, painful and tender, due to inflammationof the synovium. Furthermore, since RA is a systemic disease,inflammation can affect organs and areas of the body other than thejoints, including glands of the eyes and mouth, the lung lining, thepericardium, and blood vessels.

Traditional treatments for the management of RA and other autoimmunedisorders include fast acting “first line drugs” and slower acting“second line drugs.” The first line drugs reduce pain and inflammation.Example of such first line drugs include aspirin, naproxen, ibuprofen,etodolac and other non-steroidal anti-inflammatory drugs (NSAIDs), aswell as corticosteroids, given orally or injected directly into tissuesand joints. The second line drugs promote disease remission and preventprogressive joint destruction and are also referred to asdisease-modifying anti-rheumatic drugs or DMARDs. Examples of secondline drugs include gold, hydrochloroquine, azulfidine andimmunosuppressive agents, such as methotrexate, azathioprine,cyclophosphamide, chlorambucil and cyclosporine. Many of these drugs,however, can have detrimental side-effects. Thus, additional therapiesfor rheumatoid arthritis and other autoimmune disorders have beensought.

Tumor necrosis factor alpha (TNF-α) is a cytokine produced by numerouscell types, including monocytes and macrophages, that was originallyidentified based on its ability to induce the necrosis of certain mousetumors. Subsequently, a factor termed cachectin, associated withcachexia, was shown to be identical to TNF-α. TNF-α has been implicatedin the pathophysiology of a variety of other human diseases anddisorders, including shock, sepsis, infections, autoimmune diseases, RA,Crohn's disease, transplant rejection and graft-versus-host disease.

Because of the harmful role of human TNF-α (hTNF-α) in a variety ofhuman disorders, therapeutic strategies have been designed to inhibit orcounteract hTNF-α activity. In particular, antibodies that bind to, andneutralize, hTNF-α have been sought as a means to inhibit hTNF-αactivity. Some of the earliest of such antibodies were mouse monoclonalantibodies (mAbs), secreted by hybridomas prepared from lymphocytes ofmice immunized with hTNF-α (see, e.g., U.S. Pat. No. 5,231,024 toMoeller et al.). While these mouse anti-hTNF-α antibodies oftendisplayed high affinity for hTNF-α and were able to neutralize hTNF-αactivity, their use in vivo has been limited by problems associated withthe administration of mouse antibodies to humans, such as a short serumhalf-life, an inability to trigger certain human effector functions, andelicitation of an unwanted immune response against the mouse antibody ina human (the “human anti-mouse antibody” (HAMA) reaction).

More recently, biological therapies have been applied to the treatmentof autoimmune disorders such as rheumatoid arthritis. For example, fourTNFα inhibitors, REMICADE™ (infliximab), a chimeric anti-TNFα mAb,ENBREL™ (etanercept), a TNFR-Ig Fc fusion protein, HUMIRA™ (adalimumab),a human anti-TNFα mAb, and CIMZIA® (certolizumab pegol), a PEGylated Fabfragment, have been approved by the FDA for treatment of rheumatoidarthritis. CIMZIA® is also used for the treatment of moderate to severeCrohn's disease (CD). While such biologic therapies have demonstratedsuccess in the treatment of rheumatoid arthritis and other autoimmunedisorders such as CD, not all subjects treated respond, or respond well,to such therapy. Moreover, administration of TNFα inhibitors can inducean immune response to the drug and lead to the production ofautoantibodies such as human anti-chimeric antibodies (HACA), humananti-humanized antibodies (HAHA), and human anti-mouse antibodies(HAMA). Such HACA, HAHA, or HAMA immune responses can be associated withhypersensitive reactions and dramatic changes in pharmacokinetics andbiodistribution of the immunotherapeutic TNFα inhibitor that precludefurther treatment with the drug. Thus, there is a need in the art forassays to detect the presence of autoantibodies to biologic agents suchas anti-TNFα drugs in a patient sample to monitor biologic therapy andto guide treatment decisions. The present invention satisfies this needand provides related advantages as well.

BRIEF SUMMARY OF THE INVENTION

The present invention provides assays for detecting and measuring thepresence or level of neutralizing and non-neutralizing autoantibodies tobiologics such as anti-TNFα drug therapeutics in a sample. The presentinvention is useful for monitoring the formation of neutralizing and/ornon-neutralizing anti-drug antibodies over time while a subject is onbiologic therapy (e.g., anti-TNFα drug therapy). The present inventionis also useful for predicting and/or determining the cross-reactivity ofneutralizing anti-drug antibodies in a subject's sample with alternativebiologic therapies (e.g., alternative anti-TNFα therapies). As such, thepresent invention provides information for guiding treatment decisionsfor those subjects receiving therapy with a biologic agent and improvesthe accuracy of optimizing therapy, reducing toxicity, and/or monitoringthe efficacy of therapeutic treatment to biologic therapy.

In one aspect, the present invention provides a method for detecting thepresence of a neutralizing and/or non-neutralizing form of anautoantibody to a biologic in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled biologic and a labeled        biologic binding moiety to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic and the autoantibody            (i.e., wherein the components of the first labeled complex            are not covalently attached to each other); and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic, the labeled biologic            binding moiety, and the autoantibody (i.e., wherein the            components of the second labeled complex are not covalently            attached to each other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled biologic binding moiety,        free labeled biologic, and/or a complex of labeled biologic and        labeled biologic binding moiety;    -   (c) measuring the level of free labeled biologic binding moiety        after size exclusion chromatography (e.g., by measuring the area        under the curve (AUC) of the free labeled biologic binding        moiety peak following size exclusion chromatography (SEC)); and    -   (d) comparing the level of the free labeled biologic binding        moiety measured in step (c) to the level of free labeled        biologic binding moiety in a control sample (e.g., by measuring        the AUC of the free labeled biologic binding moiety peak        following SEC of a reference sample containing only free labeled        biologic binding moiety), thereby detecting the presence of a        neutralizing and/or non-neutralizing form of the autoantibody.

In certain embodiments, a neutralizing form of the autoantibody isdetected when the level of the free labeled biologic binding moietymeasured in step (c) is the same or substantially the same as the levelof the free labeled biologic binding moiety in the control sample. Incertain other embodiments, a non-neutralizing form of the autoantibodyis detected when the level of the free labeled biologic binding moietymeasured in step (c) is decreased (e.g., substantially decreased) orabsent (e.g., undetectable) compared to the level of the free labeledbiologic binding moiety in the control sample.

In another aspect, the present invention provides a method for measuringthe level or percent of a neutralizing form of an autoantibody to abiologic in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled biologic and a labeled        biologic binding moiety to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic and the autoantibody            (i.e., wherein the components of the first labeled complex            are not covalently attached to each other); and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic, the labeled biologic            binding moiety, and the autoantibody (i.e., wherein the            components of the second labeled complex are not covalently            attached to each other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled biologic binding moiety,        free labeled biologic, and/or a complex of labeled biologic and        labeled biologic binding moiety;    -   (c) measuring the level of free labeled biologic binding moiety        after size exclusion chromatography (e.g., by measuring the area        under the curve (AUC) of the free labeled biologic binding        moiety peak following size exclusion chromatography (SEC)); and    -   (d) comparing the level of free labeled biologic binding moiety        measured in step (c) to a normalized level or percent of free        labeled biologic binding moiety in a control sample (e.g., by        measuring and normalizing the AUC of the free labeled biologic        binding moiety peak following SEC of a reference sample        containing only free labeled biologic binding moiety to        calculate the level or percent of free labeled biologic binding        moiety), wherein the normalized level or percent of the free        labeled biologic binding moiety in the control sample        corresponds to the level or percent of a neutralizing form of        the autoantibody.

In some embodiments, the difference between the normalized level orpercent of the free labeled biologic binding moiety in the controlsample and the level of free labeled biologic binding moiety measured instep (c) corresponds to the level or percent of a non-neutralizing formof the autoantibody.

In yet another aspect, the present invention provides a method fordetermining whether a neutralizing form of an autoantibody to a firstbiologic is cross-reactive with a second (i.e., different) biologic, themethod comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of the autoantibody in a sample in accordance        with an assay described herein to determine whether the sample        is positive or negative for the neutralizing form of the        autoantibody; and

if the sample is positive for the neutralizing form of the autoantibody,then:

-   -   (b) contacting the sample with a labeled second biologic to form        a labeled complex of the labeled second biologic and the        neutralizing form of the autoantibody (i.e., wherein the        components of the labeled complex are not covalently attached to        each other);    -   (c) subjecting the labeled complex to size exclusion        chromatography to separate the labeled complex (e.g., from free        labeled second biologic); and    -   (d) detecting the labeled complex, thereby determining whether a        neutralizing form of an autoantibody to a first biologic is        cross-reactive with a second biologic.

In certain embodiments, the presence of the labeled complex is anindication that the neutralizing autoantibody against the first biologicis cross-reactive with the second biologic, i.e., the neutralizingautoantibody will inhibit the activity of both the first and secondbiological drugs.

In certain other embodiments, the absence of the labeled complex is anindication that the neutralizing autoantibody against the first biologicis not cross-reactive with the second biologic, i.e., the neutralizingautoantibody will not inhibit the activity of the second biologicaldrug.

In some embodiments, the biologic includes antibodies (e.g., anti-TNFαmonoclonal antibodies), antibody fragments, proteins (e.g., cytokinessuch as interleukins), polypeptides, peptides, fusion proteins,multivalent binding proteins, antibody-drug conjugates, vaccines,nucleic acids, sugars, recombinant forms thereof, engineered formsthereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving biologic therapy. In preferredembodiments, the sample is serum. In particular embodiments, the subjecthas a disease or disorder such as, e.g., an autoimmune disease (e.g.,rheumatoid arthritis), an inflammatory disease (e.g., inflammatory boweldisease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)),or cancer.

In certain embodiments, the sample has or is suspected of having anautoantibody to the biologic. In other embodiments, the biologicautoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In certain aspects, the assay methods of the present invention furthercomprise an acid dissociation step comprising contacting a sample withan acid prior to, during, and/or after contacting the sample with alabeled biologic and a labeled biologic binding moiety.

In certain other aspects, the assay methods of the present inventioncomprise detecting the presence or level of one or more isotypes of aneutralizing and/or non-neutralizing form of an autoantibody to abiologic in a sample.

In one particular aspect, the present invention provides a method fordetecting the presence of a neutralizing and/or non-neutralizing form ofan autoantibody to an anti-TNFα drug in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled anti-TNFα drug and a        labeled TNFα to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug and the            autoantibody (i.e., wherein the components of the first            labeled complex are not covalently attached to each other);            and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug, the labeled TNFα,            and the autoantibody (i.e., wherein the components of the            second labeled complex are not covalently attached to each            other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled TNFα, free labeled        anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and        labeled TNFα;    -   (c) measuring the level of free labeled TNFα after size        exclusion chromatography (e.g., by measuring the area under the        curve (AUC) of the free labeled TNFα peak following size        exclusion chromatography (SEC)); and    -   (d) comparing the level of the free labeled TNFα measured in        step (c) to the level of free labeled TNFα in a control sample        (e.g., by measuring the AUC of the free labeled TNFα peak        following SEC of a reference sample containing only free labeled        TNFα), thereby detecting the presence of a neutralizing and/or        non-neutralizing form of the autoantibody.

In certain embodiments, a neutralizing form of the autoantibody isdetected when the level of the free labeled TNFα measured in step (c) isthe same or substantially the same as the level of the free labeled TNFαin the control sample. In certain other embodiments, a non-neutralizingform of the autoantibody is detected when the level of the free labeledTNFα measured in step (c) is decreased (e.g., substantially decreased)or absent (e.g., undetectable) compared to the level of the free labeledTNFα in the control sample.

In another particular aspect, the present invention provides a methodfor measuring the level or percent of a neutralizing form of anautoantibody to an anti-TNFα drug in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled anti-TNFα drug and a        labeled TNFα to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug and the            autoantibody (i.e., wherein the components of the first            labeled complex are not covalently attached to each other);            and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug, the labeled TNFα,            and the autoantibody (i.e., wherein the components of the            second labeled complex are not covalently attached to each            other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled TNFα, free labeled        anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and        labeled TNFα;    -   (c) measuring the level of free labeled TNFα after size        exclusion chromatography (e.g., by measuring the area under the        curve (AUC) of the free labeled TNFα peak following size        exclusion chromatography (SEC)); and    -   (d) comparing the level of free labeled TNFα measured in        step (c) to a normalized level or percent of free labeled TNFα        in a control sample (e.g., by measuring and normalizing the AUC        of the free labeled TNFα peak following SEC of a reference        sample containing only free labeled TNFα to calculate the level        or percent of free labeled TNFα), wherein the normalized level        or percent of the free labeled TNFα in the control sample        corresponds to the level or percent of a neutralizing form of        the autoantibody.

In some embodiments, the difference between the normalized level orpercent of the free labeled TNFα in the control sample and the level offree labeled TNFα measured in step (c) corresponds to the level orpercent of a non-neutralizing form of the autoantibody.

In yet another particular aspect, the present invention provides amethod for determining whether a neutralizing form of an autoantibody toa first anti-TNFα drug is cross-reactive with a second (i.e., different)anti-TNFα drug, the method comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of the autoantibody in a sample in accordance        with an assay described herein to determine whether the sample        is positive or negative for the neutralizing form of the        autoantibody; and

if the sample is positive for the neutralizing form of the autoantibody,then:

-   -   (b) contacting the sample with a labeled second anti-TNFα drug        to form a labeled complex of the labeled second anti-TNFα drug        and the neutralizing form of the autoantibody (i.e., wherein the        components of the labeled complex are not covalently attached to        each other);    -   (c) subjecting the labeled complex to size exclusion        chromatography to separate the labeled complex (e.g., from free        labeled second anti-TNFα drug); and    -   (d) detecting the labeled complex, thereby determining whether a        neutralizing form of an autoantibody to a first anti-TNFα drug        is cross-reactive with a second anti-TNFα drug.

In certain embodiments, the presence of the labeled complex is anindication that the neutralizing autoantibody against the firstanti-TNFα drug is cross-reactive with the second anti-TNFα drug, i.e.,the neutralizing autoantibody will inhibit the activity of both thefirst and second anti-TNFα drugs.

In certain other embodiments, the absence of the labeled complex is anindication that the neutralizing autoantibody against the firstanti-TNFα drug is not cross-reactive with the second anti-TNFα drug,i.e., the neutralizing autoantibody will not inhibit the activity of thesecond anti-TNFα drug.

In some embodiments, the anti-TNFα drug is selected from the groupconsisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™(adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving anti-TNFα drug therapy. Inpreferred embodiments, the sample is serum. In particular embodiments,the subject has a TNFα-mediated disease or disorder such as, e.g., anautoimmune disease (e.g., rheumatoid arthritis) or an inflammatorydisease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease(CD) or ulcerative colitis (UC)).

In certain embodiments, the sample has or is suspected of having anautoantibody to the anti-TNFα drug. In other embodiments, the anti-TNFαdrug autoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In certain aspects, the assay methods of the present invention furthercomprise an acid dissociation step comprising contacting a sample withan acid prior to, during, and/or after contacting the sample with alabeled anti-TNFα drug and a labeled TNFα.

In certain other aspects, the assay methods of the present inventioncomprise detecting the presence or level of one or more isotypes of aneutralizing and/or non-neutralizing form of an autoantibody to ananti-TNFα drug in a sample.

In a further aspect, the present invention provides a method formonitoring and/or optimizing therapy to a biologic in a subjectreceiving a course of therapy with the biologic, the method comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of an autoantibody to the biologic in        accordance with the assay described herein at a plurality of        time points over the course of therapy;    -   (b) detecting a change in the presence, level, or percent of the        neutralizing form of the autoantibody over time; and    -   (c) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the        presence, level, or percent of the neutralizing form of the        autoantibody over time.

In one particular aspect, the present invention provides a method formonitoring and/or optimizing therapy to a biologic in a subjectreceiving a course of therapy with the biologic, the method comprising:

-   -   (a) measuring the level or percent of a neutralizing form of an        autoantibody to the biologic in a first sample from the subject        as described herein at time point t₀;    -   (b) measuring the level or percent of the neutralizing form of        the autoantibody in a second sample from the subject as        described herein at time point t₁;    -   (c) optionally repeating step (b) with n additional samples from        the subject at time points t_(n+1), wherein n is an integer from        1 to about 25 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or any        range therein);    -   (d) detecting a change in the level or percent of the        neutralizing form of the autoantibody from time points t₀ to t₁        or from time points t₀ to t_(n+1); and    -   (e) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the level        or percent of the neutralizing form of the autoantibody over        time.

In an additional aspect, the present invention provides a method foroptimizing therapy and/or reducing toxicity in a subject receiving acourse of therapy with a first biologic, the method comprising:

-   -   (a) determining whether a neutralizing form of an autoantibody        to the first biologic is cross-reactive with a second (i.e.,        different) biologic by detecting or measuring the presence,        level, or percent of a neutralizing form of the autoantibody in        a sample from the subject in accordance with an assay described        herein; and    -   (b) determining that a different course of therapy should be        administered to the subject if the neutralizing form of the        autoantibody is cross-reactive with the second biologic.

In one particular aspect, the present invention provides a method formonitoring and/or optimizing therapy to an anti-TNFα drug in a subjectreceiving a course of therapy with the anti-TNFα drug, the methodcomprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of an autoantibody to the anti-TNFα drug in        accordance with the assay described herein at a plurality of        time points over the course of therapy;    -   (b) detecting a change in the presence, level, or percent of the        neutralizing form of the autoantibody over time; and    -   (c) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the        presence, level, or percent of the neutralizing form of the        autoantibody over time.

In another particular aspect, the present invention provides a methodfor monitoring and/or optimizing therapy to an anti-TNFα drug in asubject receiving a course of therapy with the anti-TNFα drug, themethod comprising:

-   -   (a) measuring the level or percent of a neutralizing form of an        autoantibody to the anti-TNFα drug in a first sample from the        subject as described herein at time point t₀;    -   (b) measuring the level or percent of the neutralizing form of        the autoantibody in a second sample from the subject as        described herein at time point t₁;    -   (c) optionally repeating step (b) with n additional samples from        the subject at time points t_(n+1), wherein n is an integer from        1 to about 25 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or any        range therein);    -   (d) detecting a change in the level or percent of the        neutralizing form of the autoantibody from time points t₀ to t₁        or from time points t₀ to t_(n+1); and    -   (e) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the level        or percent of the neutralizing form of the autoantibody over        time.

In yet another particular aspect, the present invention provides amethod for optimizing therapy and/or reducing toxicity in a subjectreceiving a course of therapy with a first anti-TNFα drug, the methodcomprising:

-   -   (a) determining whether a neutralizing form of an autoantibody        to the first anti-TNFα drug is cross-reactive with a second        (i.e., different) anti-TNFα drug by detecting or measuring the        presence, level, or percent of a neutralizing form of the        autoantibody in a sample from the subject in accordance with an        assay described herein; and    -   (b) determining that a different course of therapy should be        administered to the subject if the neutralizing form of the        autoantibody is cross-reactive with the second anti-TNFα drug.

Other objects, features, and advantages of the present invention will beapparent to one of skill in the art from the following detaileddescription and figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates that there was a clear relationship between NAbpercent (y-axis) and ATI levels.

FIG. 2 illustrates that an ATI concentration ≧60 U/ml is predictive ofNAb+.

FIG. 3 illustrates that ATI predicts NAb with a ROC AUC of 0.931.

FIG. 4 illustrates detection of ATI by the fluid phase mobility shiftassay of the present invention.

FIG. 5 illustrates an exemplary ATI/IFX fluid phase mobility shift assayof the present invention.

FIG. 6 illustrates a non-neutralizing anti-drug antibody (ADA) assay ofthe present invention.

FIG. 7 illustrates a neutralizing ADA assay of the present invention.

FIG. 8 illustrates the levels of IFX and ATI over a time course of 5samples from a UC patient taken 1, 2, or 3 months apart.

FIG. 9 shows peak analysis to determine the percentage of free TNFα overtime in a UC patient.

FIG. 10 illustrates a shift from the presence of non-neutralizingautoantibodies to neutralizing autoantibodies over time as exemplifiedin 3 samples from a UC patient taken 2 or 3 months apart and spiked withIFX.

FIG. 11 shows peak analysis to determine the percentage of free TNFαover time in samples from a UC patient that were spiked with IFX.

FIG. 12 shows the use of rabbit anti-human IgG1 Fc as a non-neutralizingantibody (Ab) control.

FIG. 13 shows the use of ATI positive serum as a mixed neutralizingantibody (NAb)/non-neutralizing antibody (Ab) control.

FIG. 14 shows that purification of ATI from ATI positive serum resultsin loss of weaker affinity NAb.

FIG. 15 illustrates peak analysis from a UC patient case study todetermine the percentage of free TNFα in these various controls.

FIG. 16 shows a peak analysis from a CD patient case study to determinethe percentage of free TNFα over a time course of 4 samples taken 7 or 8weeks apart during a 30-week period.

FIG. 17 shows a peak analysis from another CD patient case study todetermine the percentage of free TNFα over a time course of 3 samplestaken during a 50-week period.

FIG. 18 shows a peak analysis from 4 additional CD patient case studiesto determine the percentage of free TNFα in a sample at a particularweek during or after induction or maintenance of therapy.

FIG. 19 shows detection of non-neutralizing antibody activity via themobility shift assay.

FIG. 20 depicts the cross-reactivity of ADA against both IFX and ADL,wherein the binding site of ADA mimics the binding site of TNFα and cantherefore bind to multiple anti-TNF drugs.

FIG. 21 shows two patient examples (Patients 1 and 2) in whichcross-reactivity of NAb produced in response to one anti-TNF drug wasdetermined for other anti-TNF drugs. In particular, NAb which developedwhen the patient was on Remicade (IFX) were tested against Humira (ADL).

FIG. 22 shows exemplary embodiments of the assays of the presentinvention to detect the presence of non-neutralizing antibodies(non-NAb) (top) or neutralizing antibodies (NAb) (bottom) against a drugsuch as IFX or ADL.

FIG. 23 shows the generation and use of a NAb standard curve.

FIG. 24 provides the results of a case study for Patient 3, who wastreated with IFX but lost response to IFX, to determine thecross-reactivity of NAb generated against IFX to ADL.

FIG. 25 provides the results of a case study for Patient 4, who wastreated with IFX but lost response to IFX, to determine thecross-reactivity of NAb generated against IFX to ADL.

FIG. 26 shows non-limiting examples of patient studies which demonstrateATI affinity maturation and the development of cross-reactive ATI.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction

The present invention is based in part on the discovery that ahomogeneous mobility shift assay using size exclusion chromatography andoptionally acid dissociation to enable equilibration of immune complexesis particularly advantageous for measuring the presence or level ofneutralizing and non-neutralizing forms of autoantibodies (e.g., HACA,HAHA, etc.) that are generated against biologics such as anti-TNFαdrugs. Such autoantibodies are also known as anti-drug antibodies orADA, and neutralizing and non-neutralizing forms thereof are also knownas NAb and non-NAb, respectively.

In particular embodiments, the homogeneous mobility shift assays of theinvention are performed by contacting a subject's sample with (e.g.,fluorescently) labeled biologic (e.g., anti-TNFα drug) and (e.g.,fluorescently) labeled biologic binding moiety (e.g., TNFα). The assaysdescribed herein are advantageous for at least the following reasons:they obviate the need for wash steps which remove low affinity ADA; theyuse distinct labels such as fluorophores that allow for detection on thevisible, IR, and/or near IR (NIR) spectra which decreases background andserum interference issues; they increase the ability to detectneutralizing and/or non-neutralizing ADA in subjects with a low titerdue to the high sensitivity of fluorescent label detection; and theyoccur as a liquid phase reaction, thereby reducing the chance of anychanges in the epitope by attachment to a solid surface such as an ELISAplate.

In exemplary embodiments, the assays of the present invention areadvantageous because they enable time course case studies of IBDsubjects on anti-TNFα drug therapy for monitoring the formation ofneutralizing and/or non-neutralizing anti-drug antibodies in multiplesamples at different time points. The assays of the present inventionare also advantageous because they enable the determination of whetherthere is a shift from non-neutralizing to neutralizing anti-drugantibodies over time while a subject is on anti-TNFα drug therapy.Without being bound to any particular theory, neutralizing anti-drugantibodies may have significant negative clinical consequences becausethey interfere with the binding between the anti-TNFα drug and TNFα,thereby inducing a loss of efficacy.

In additional exemplary embodiments, the assays of the present inventionfind utility in predicting and/or determining the cross-reactivity ofneutralizing anti-drug antibodies in a subject's sample with alternativebiological drugs such as other anti-TNF drugs. For illustration purposesonly, if the sample contains neutralizing ADA to one anti-TNFα drug,these neutralizing ADA will likely cross-react and be neutralizing toother anti-TNFα drugs, such that the recommended treatment adjustmentfor the subject would be to switch to a drug with a different mechanismof action (e.g., a non-anti-TNF agent). However, if the sample containsnon-neutralizing ADA to one anti-TNFα drug, the recommended treatmentadjustment for the subject could be to switch to another anti-TNFα drug.

Accordingly, the present invention addresses and overcomes currentlimitations associated with the administration of anti-TNFα drugs, suchas infliximab and adalimumab, in part, by providing information usefulfor guiding treatment decisions for those subjects receiving anti-TNFαdrug therapy. The methods of the present invention are particularlyuseful for monitoring those subjects receiving an anti-TNFα drug todetect or measure the formation and/or development of neutralizing ADA(e.g., over time during a course of anti-TNFα drug therapy) and are alsouseful to detect or measure a change in (e.g., increase) the amount,percent, or ratio of neutralizing ADA compared to non-neutralizing ADAover time while a subject is on anti-TNFα drug therapy.

As such, the present invention provides methods for determining whenand/or how (1) to adjust or modify (e.g., increase or decrease) thesubsequent dose of an anti-TNFα drug to optimize therapeutic efficacyand/or to reduce toxicity in view of the presence, level, or percent ofneutralizing ADA, (2) to combine an anti-TNFα drug (e.g., at an initial,increased, decreased, or same dose) with one or more immunosuppressiveagents such as methotrexate (MTX) or azathioprine (AZA) in view of thepresence, level, or percent of neutralizing ADA, and/or (3) to changethe current course of therapy (e.g., switch to a different anti-TNFαdrug or to a drug that targets a different mechanism) in view of thepresence, level, or percent of neutralizing ADA. Such methods are usefulfor ensuring that subjects receiving anti-TNFα drugs are getting theright dose, that they are not developing an immune response against thedrug, and that they should be switched to a different drug due tofailure with the initial drug (e.g., development of cross-reactiveneutralizing ADA against the initial anti-TNFα drug).

II. Definitions

As used herein, the following terms have the meanings ascribed to themunless specified otherwise.

The terms “biologic” or “biologic agent” or “biological drug” as usedherein encompass products and substances produced from or extracted froma biological system (e.g., a living organism). Non-limiting examples ofbiologics include antibodies, antibody fragments, proteins,polypeptides, peptides, fusion proteins (e.g., Ig fusion proteins or Fcfusion proteins), multivalent binding proteins (e.g., DVD Ig),antibody-drug conjugates, vaccines, nucleic acids, sugars, recombinantforms thereof, engineered forms thereof, and combinations thereof.

The term “biologic binding moiety” includes any molecule, agent, orsubstance that (e.g., specifically) binds to or interacts with abiologic. In certain instances, a neutralizing form of the autoantibodyinterferes with the binding between the biologic binding moiety and thebiologic. In certain other instances, a non-neutralizing form of theautoantibody does not interfere with the binding between the biologicbinding moiety and the biologic. As one non-limiting example, thebiologic binding moiety comprises TNFα when the biologic comprises ananti-TNFα drug. As another non-limiting example, the biologic bindingmoiety comprises an interleukin receptor (e.g., a soluble extracellularfragment of an interleukin receptor) when the biologic comprises aninterleukin such as IL-2.

The terms “anti-TNFα drug” or “TNFα inhibitor” as used herein areintended to encompass agents including proteins, antibodies, antibodyfragments, fusion proteins (e.g., Ig fusion proteins or Fc fusionproteins), multivalent binding proteins (e.g., DVD Ig), small moleculeTNFα antagonists and similar naturally- or nonnaturally-occurringmolecules, and/or recombinant and/or engineered forms thereof, that,directly or indirectly, inhibit TNFα activity, such as by inhibitinginteraction of TNFα with a cell surface receptor for TNFα, inhibitingTNFα protein production, inhibiting TNFα gene expression, inhibitingTNFα secretion from cells, inhibiting TNFα receptor signaling or anyother means resulting in decreased TNFα activity in a subject. The term“anti-TNFα drug” or “TNFα inhibitor” preferably includes agents whichinterfere with TNFα activity. Examples of anti-TNFα drugs include,without limitation, infliximab (REMICADE™, Johnson and Johnson), humananti-TNF monoclonal antibody adalimumab (D2E7/HUMIRA™, AbbottLaboratories), etanercept (ENBREL™, Amgen), certolizumab pegol (CIMZIA®,UCB, Inc.), golimumab (SIMPONI®; CNTO 148), CDP 571 (Celltech), CDP 870(Celltech), as well as other compounds which inhibit TNFα activity, suchthat when administered to a subject suffering from or at risk ofsuffering from a disorder in which TNFα activity is detrimental (e.g.,RA), the disorder is treated.

The term “TNFα” is intended to include a human cytokine that exists as a17 kDa secreted form and a 26 kDa membrane associated form, thebiologically active form of which is composed of a trimer ofnoncovalently bound 17 kDa molecules. The structure of TNFα is describedfurther in, for example, Jones et al., Nature, 338:225-228 (1989). Theterm TNFα is intended to include human TNFα, a recombinant human TNFα(rhTNF-α), or TNFα that is at least about 80% identity to the human TNFαprotein. Human TNFα consists of a 35 amino acid (aa) cytoplasmic domain,a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD)(Pennica, D. et al. (1984) Nature 312:724). Within the ECD, human TNFαshares 97% aa sequence identity with rhesus TNFα, and 71% to 92% aasequence identity with bovine, canine, cotton rat, equine, feline,mouse, porcine, and rat TNFα. TNFα can be prepared by standardrecombinant expression methods or purchased commercially (R & D Systems,Catalog No. 210-TA, Minneapolis, Minn.).

In certain embodiments, “TNFα” is an “antigen,” which includes amolecule or a portion of the molecule capable of being bound by ananti-TNF-α drug. TNFα can have one or more than one epitope. In certaininstances, TNFα will react, in a highly selective manner, with ananti-TNFα antibody. Preferred antigens that bind antibodies, fragments,and regions of anti-TNFα antibodies include at least 5 amino acids ofhuman TNFα. In certain instances, TNFα is a sufficient length having anepitope of TNFα that is capable of binding anti-TNFα antibodies,fragments, and regions thereof.

The term “size exclusion chromatography” or “SEC” includes achromatographic method in which molecules in solution are separatedbased on their size and/or hydrodynamic volume. It is applied to largemolecules or macromolecular complexes such as proteins and theirconjugates. Typically, when an aqueous solution is used to transport thesample through the column, the technique is known as gel filtrationchromatography.

The terms “complex,” “immuno-complex,” “conjugate,” and“immunoconjugate” include, but are not limited to, TNFα bound (e.g., bynon-covalent means) to an anti-TNFα drug, an anti-TNFα drug bound (e.g.,by non-covalent means) to an autoantibody against the anti-TNFα drug(e.g., a neutralizing or non-neutralizing anti-drug antibody), and ananti-TNFα drug bound (e.g., by non-covalent means) to both TNFα and anautoantibody against the anti-TNFα drug (e.g., a neutralizing ornon-neutralizing anti-drug antibody).

As used herein, an entity that is modified by the term “labeled”includes any entity, molecule, protein, enzyme, antibody, antibodyfragment, cytokine, or related species that is conjugated with anothermolecule or chemical entity that is empirically detectable. Chemicalspecies suitable as labels for labeled-entities include, but are notlimited to, fluorescent dyes, e.g. Alexa Fluor® dyes such as AlexaFluor® 647, quantum dots, optical dyes, luminescent dyes, andradionuclides, e.g. ¹²⁵I.

The phrase “fluorescence label detection” includes a means for detectinga fluorescent label. Means for detection include, but are not limitedto, a spectrometer, a fluorimeter, a photometer, and a detection devicecommonly incorporated with a chromatography instrument such as, but notlimited to, size exclusion-high performance liquid chromatography, suchas, but not limited to, an Agilent-1200 HPLC System.

The phrase “optimize therapy” includes optimizing the dose (e.g., theeffective amount or level) and/or the type of a particular therapy. Forexample, optimizing the dose of an anti-TNFα drug includes increasing ordecreasing the amount of the anti-TNFα drug subsequently administered toa subject. In certain instances, optimizing the type of an anti-TNFαdrug includes changing the administered anti-TNFα drug from one drug toa different drug (e.g., a different anti-TNFα drug or a drug thattargets a different mechanism). In other instances, optimizing therapyincludes co-administering a dose of an anti-TNFα drug (e.g., at anincreased, decreased, or same dose as the previous dose) in combinationwith one or more immunosuppressive drugs.

The term “co-administer” includes to administer more than one activeagent, such that the duration of physiological effect of one activeagent overlaps with the physiological effect of a second active agent.

The term “subject,” “patient,” or “individual” typically includeshumans, but also includes other animals such as, e.g., other primates,rodents, canines, felines, equines, ovines, porcines, and the like.

The term “course of therapy” includes any therapeutic approach taken torelieve or prevent one or more symptoms associated with a disease ordisorder. The term encompasses administering any compound, drug,procedure, and/or regimen useful for improving the health of anindividual with a disease or disorder and includes any of thetherapeutic agents described herein. As a non-limiting example, thecourse of therapy or the dose of the current course of therapy can bechanged (e.g., increased or decreased) based upon the presence orconcentration level of TNFα, anti-TNFα drug, and/or anti-drug antibody(e.g., the presence, level, or percent of neutralizing and/ornon-neutralizing anti-drug antibody determined using the methods of theinvention).

The term “immunosuppressive drug” or “immunosuppressive agent” includesany substance capable of producing an immunosuppressive effect, e.g.,the prevention or diminution of the immune response, as by irradiationor by administration of drugs such as anti-metabolites, anti-lymphocytesera, antibodies, etc. Examples of immunosuppressive drugs include,without limitation, thiopurine drugs such as azathioprine (AZA) andmetabolites thereof; anti-metabolites such as methotrexate (MTX);sirolimus (rapamycin); temsirolimus; everolimus; tacrolimus (FK-506);FK-778; anti-lymphocyte globulin antibodies, anti-thymocyte globulinantibodies, anti-CD3 antibodies, anti-CD4 antibodies, and antibody-toxinconjugates; cyclosporine; mycophenolate; mizoribine monophosphate;scoparone; glatiramer acetate; metabolites thereof; pharmaceuticallyacceptable salts thereof; derivatives thereof; prodrugs thereof; andcombinations thereof.

The term “thiopurine drug” includes azathioprine (AZA), 6-mercaptopurine(6-MP), or any metabolite thereof that has therapeutic efficacy andincludes, without limitation, 6-thioguanine (6-TG),6-methylmercaptopurine riboside, 6-thioinosine nucleotides (e.g.,6-thioinosine monophosphate, 6-thioinosine diphosphate, 6-thioinosinetriphosphate), 6-thioguanine nucleotides (e.g., 6-thioguanosinemonophosphate, 6-thioguanosine diphosphate, 6-thioguanosinetriphosphate), 6-thioxanthosine nucleotides (e.g., 6-thioxanthosinemonophosphate, 6-thioxanthosine diphosphate, 6-thioxanthosinetriphosphate), derivatives thereof, analogues thereof, and combinationsthereof.

The term “sample” includes any biological specimen obtained from anindividual. Samples include, without limitation, whole blood, plasma,serum, red blood cells, white blood cells (e.g., peripheral bloodmononuclear cells (PBMC), polymorphonuclear (PMN) cells), ductal lavagefluid, nipple aspirate, lymph (e.g., disseminated tumor cells of thelymph node), bone marrow aspirate, saliva, urine, stool (i.e., feces),sputum, bronchial lavage fluid, tears, fine needle aspirate (e.g.,harvested by random periareolar fine needle aspiration), any otherbodily fluid, a tissue sample such as a biopsy of a site of inflammation(e.g., needle biopsy), cellular extracts thereof, and an immunoglobulinenriched fraction derived from one or more of these bodily fluids ortissues. In some embodiments, the sample is whole blood, a fractionalcomponent thereof such as plasma, serum, or a cell pellet, or animmunoglobulin enriched fraction thereof. One skilled in the art willappreciate that samples such as serum samples can be diluted prior tothe analysis. In certain embodiments, the sample is obtained byisolating PBMCs and/or PMN cells using any technique known in the art.In certain other embodiments, the sample is a tissue biopsy such as,e.g., from a site of inflammation such as a portion of thegastrointestinal tract or synovial tissue.

The steps of the methods of the present invention do not necessarilyhave to be performed in the particular order in which they arepresented. A person of ordinary skill in the art would understand thatother orderings of the steps of the methods of the invention areencompassed within the scope of the present invention.

Brackets, “[ ]” indicate that the species within the brackets arereferred to by their concentration.

III. Description of the Embodiments

The present invention provides assays for detecting and measuring thepresence or level of neutralizing and non-neutralizing autoantibodies tobiologics such as anti-TNFα drug therapeutics in a sample. The presentinvention is useful for monitoring the formation of neutralizing and/ornon-neutralizing anti-drug antibodies over time while a subject is onbiologic therapy (e.g., anti-TNFα drug therapy). The present inventionis also useful for predicting and/or determining the cross-reactivity ofneutralizing anti-drug antibodies in a subject's sample with alternativebiologic therapies (e.g., alternative anti-TNFα therapies). As such, thepresent invention provides information for guiding treatment decisionsfor those subjects receiving therapy with a biologic agent and improvesthe accuracy of optimizing therapy, reducing toxicity, and/or monitoringthe efficacy of therapeutic treatment to biologic therapy.

In one aspect, the present invention provides a method for detecting thepresence of a neutralizing and/or non-neutralizing form of anautoantibody to a biologic in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled biologic and a labeled        biologic binding moiety to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic and the autoantibody            (i.e., wherein the components of the first labeled complex            are not covalently attached to each other); and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic, the labeled biologic            binding moiety, and the autoantibody (i.e., wherein the            components of the second labeled complex are not covalently            attached to each other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled biologic binding moiety,        free labeled biologic, and/or a complex of labeled biologic and        labeled biologic binding moiety;    -   (c) measuring the level of free labeled biologic binding moiety        after size exclusion chromatography (e.g., by measuring the area        under the curve (AUC) of the free labeled biologic binding        moiety peak following size exclusion chromatography (SEC)); and    -   (d) comparing the level of the free labeled biologic binding        moiety measured in step (c) to the level of free labeled        biologic binding moiety in a control sample (e.g., by measuring        the AUC of the free labeled biologic binding moiety peak        following SEC of a reference sample containing only free labeled        biologic binding moiety), thereby detecting the presence of a        neutralizing and/or non-neutralizing form of the autoantibody.

In some embodiments, a neutralizing form of the autoantibody interfereswith the binding between the biologic and biologic binding moiety. Inother embodiments, a non-neutralizing form of the autoantibody does notinterfere with the binding between the biologic and biologic bindingmoiety.

In some instances, free labeled biologic binding moiety consists oflabeled biologic binding moiety that is substantially free of boundbiologic (e.g., labeled and/or unlabeled biologic).

In certain embodiments, a neutralizing form of the autoantibody isdetected when the level of the free labeled biologic binding moietymeasured in step (c) is the same or substantially the same as the levelof the free labeled biologic binding moiety in the control sample. Incertain other embodiments, a non-neutralizing form of the autoantibodyis detected when the level of the free labeled biologic binding moietymeasured in step (c) is decreased (e.g., substantially decreased) orabsent (e.g., undetectable) compared to the level of the free labeledbiologic binding moiety in the control sample.

In particular embodiments, the level of the free labeled biologicbinding moiety measured in step (c) is considered to be substantiallythe same as the level of the free labeled biologic binding moiety in thecontrol sample when it is at least about 70%, 75%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% the level of the free labeled biologic binding moietymeasured in the control sample. In particular embodiments, the level ofthe free labeled biologic binding moiety measured in step (c) isconsidered to be substantially decreased compared to the level of thefree labeled biologic binding moiety in the control sample when it is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% lessthan the level of the free labeled biologic binding moiety measured inthe control sample.

In some embodiments, the level of free labeled biologic binding moietyis measured by integrating the area under the curve (AUC) of the freelabeled biologic binding moiety peak from a plot of signal intensity asa function of elution time from the size exclusion chromatography (e.g.,SEC-HPLC).

In some embodiments, the biologic includes antibodies (e.g., anti-TNFαmonoclonal antibodies), antibody fragments, proteins (e.g., cytokinessuch as interleukins), polypeptides, peptides, fusion proteins,multivalent binding proteins, antibody-drug conjugates, vaccines,nucleic acids, sugars, recombinant forms thereof, engineered formsthereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving biologic therapy. In preferredembodiments, the sample is serum. In particular embodiments, the subjecthas a disease or disorder such as, e.g., an autoimmune disease (e.g.,rheumatoid arthritis), an inflammatory disease (e.g., inflammatory boweldisease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)),or cancer.

In certain embodiments, the sample has or is suspected of having anautoantibody to the biologic. In other embodiments, the biologicautoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In another aspect, the present invention provides a method for measuringthe level or percent of a neutralizing form of an autoantibody to abiologic in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled biologic and a labeled        biologic binding moiety to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic and the autoantibody            (i.e., wherein the components of the first labeled complex            are not covalently attached to each other); and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled biologic, the labeled biologic            binding moiety, and the autoantibody (i.e., wherein the            components of the second labeled complex are not covalently            attached to each other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled biologic binding moiety,        free labeled biologic, and/or a complex of labeled biologic and        labeled biologic binding moiety;    -   (c) measuring the level of free labeled biologic binding moiety        after size exclusion chromatography (e.g., by measuring the area        under the curve (AUC) of the free labeled biologic binding        moiety peak following size exclusion chromatography (SEC)); and    -   (d) comparing the level of free labeled biologic binding moiety        measured in step (c) to a normalized level or percent of free        labeled biologic binding moiety in a control sample (e.g., by        measuring and normalizing the AUC of the free labeled biologic        binding moiety peak following SEC of a reference sample        containing only free labeled biologic binding moiety to        calculate the level or percent of free labeled biologic binding        moiety), wherein the normalized level or percent of the free        labeled biologic binding moiety in the control sample        corresponds to the level or percent of a neutralizing form of        the autoantibody.

In some embodiments, the difference between the normalized level orpercent of the free labeled biologic binding moiety in the controlsample and the level of free labeled biologic binding moiety measured instep (c) corresponds to the level or percent of a non-neutralizing formof the autoantibody.

In some instances, free labeled biologic binding moiety consists oflabeled biologic binding moiety that is substantially free of boundbiologic (e.g., labeled and/or unlabeled biologic).

In particular embodiments, the level or percent of the free labeledbiologic binding moiety in a control sample is normalized by measuringthe peak area (e.g., by measuring the AUC) of a complex formed betweenthe labeled biologic and the labeled biologic binding moiety (e.g.,“labeled complex”), and then subtracting the measured peak area of thelabeled complex from the peak area of the free labeled biologic bindingmoiety (e.g., by measuring the AUC of the free labeled biologic bindingmoiety peak).

In certain embodiments, the level of the free labeled biologic bindingmoiety is measured by integrating the area under the curve (AUC) of thefree labeled biologic binding moiety peak from a plot of signalintensity as a function of elution time from the size exclusionchromatography (e.g., SEC-HPLC). In other embodiments, the level of acomplex formed between the labeled biologic and labeled biologic bindingmoiety is measured by integrating the AUC of the free labeled biologicbinding moiety peak from a plot of signal intensity as a function ofelution time from the size exclusion chromatography (e.g., SEC-HPLC).

In certain embodiments, a subpopulation of the autoantibody to abiologic (e.g., ADA) is a neutralizing form of the autoantibody (e.g.,NAb). In some embodiments, the total level of an autoantibody to abiologic in a sample can be calculated by adding the levels of bothneutralizing and non-neutralizing forms of the autoantibody measured inaccordance with the methods of the invention.

In some embodiments, the level of the free labeled biologic bindingmoiety measured in step (c) is further compared to a negative control, apositive control, or a combination thereof. In further embodiments, thepercent of the neutralizing form of the autoantibody (e.g., NAb)determined in step (d) is compared to a cutoff value or reference rangeestablished from a healthy control (e.g., normal human serum). In someembodiments, the cutoff value or reference range is expressed as athreshold percent of NAb that the sample must have in order to beconsidered positive for NAb. In such embodiments, the sample is positivefor NAb when the percent of NAb determined in step (d) is greater thanor equal to the cutoff value or reference range established from thehealthy control. In other embodiments, the sample is negative for NAbwhen the percent of NAb determined in step (d) is less than the cutoffvalue or reference range established from the healthy control.Non-limiting examples of cutoff values or reference ranges include,e.g., at least about 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%, 2.50%,2.60%, 2.70%, 2.80%, 2.90%, 3.00%, 3.01%, 3.02%, 3.03%, 3.04%, 3.05%,3.06%, 3.07%, 3.08%, 3.09%, 3.10%, 3.20%, 3.30%, 3.40%, 3.50%, 4.00%,4.50%, 5.00%, 5.50%, 6.00%, 6.50%, 7.00%, 7.50%, 8.00%, 8.50%, 9.00%,9.50%, 10.00% NAb, or any range therein.

In some embodiments, all of the autoantibodies to the biologic areneutralizing antibodies and the sample is defined as having 100%neutralizing anti-drug antibodies (NAb) and/or 0% non-neutralizinganti-drug antibodies (non-NAb). In these embodiments, the level of thefree labeled biologic binding moiety measured in step (c) is generallythe same as the level of the free labeled biologic binding moiety in thecontrol sample, and the autoantibodies are predicted to completely blockor interfere with the binding between the biologic and the biologicbinding moiety.

In other embodiments, none of the autoantibodies to the biologic areneutralizing antibodies and the sample is defined as having 100% non-NAband/or 0% NAb. In these embodiments, the level of the free labeledbiologic binding moiety measured in step (c) is generally absent (e.g.,undetectable) compared to the level of the free labeled biologic bindingmoiety in the control sample, and the autoantibodies are predicted tonot completely block or interfere with the binding between the biologicand the biologic binding moiety.

In further embodiments, when both neutralizing and non-neutralizingforms of the autoantibody are present in a sample, the percent of eachspecies can be expressed on their own (e.g., 50% NAb or 50% non-NAb isdefined as an equal proportion of NAb and non-NAb in a sample) or as aratio. In certain instances, the ratio is calculated by dividing thepercent of NAb by the percent of non-NAb, or vice versa. In otherinstances, the ratio is calculated by dividing the level of NAb by thelevel of non-NAb, or vice versa.

In some embodiments, the biologic includes antibodies (e.g., anti-TNFαmonoclonal antibodies), antibody fragments, proteins (e.g., cytokinessuch as interleukins), polypeptides, peptides, fusion proteins,multivalent binding proteins, antibody-drug conjugates, vaccines,nucleic acids, sugars, recombinant forms thereof, engineered formsthereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving biologic therapy. In preferredembodiments, the sample is serum. In particular embodiments, the subjecthas a disease or disorder such as, e.g., an autoimmune disease (e.g.,rheumatoid arthritis), an inflammatory disease (e.g., inflammatory boweldisease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)),or cancer.

In certain embodiments, the sample has or is suspected of having anautoantibody to the biologic. In other embodiments, the biologicautoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In yet another aspect, the present invention provides a method fordetermining whether a neutralizing form of an autoantibody to a firstbiologic is cross-reactive with a second (i.e., different) biologic, themethod comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of the autoantibody in a sample in accordance        with an assay described herein to determine whether the sample        is positive or negative for the neutralizing form of the        autoantibody; and

if the sample is positive for the neutralizing form of the autoantibody,then:

-   -   (b) contacting the sample with a labeled second biologic to form        a labeled complex of the labeled second biologic and the        neutralizing form of the autoantibody (i.e., wherein the        components of the labeled complex are not covalently attached to        each other);    -   (c) subjecting the labeled complex to size exclusion        chromatography to separate the labeled complex (e.g., from free        labeled second biologic); and    -   (d) detecting the labeled complex, thereby determining whether a        neutralizing form of an autoantibody to a first biologic is        cross-reactive with a second biologic.

In certain embodiments, the presence of the labeled complex is anindication that the neutralizing autoantibody against the first biologicis cross-reactive with the second biologic, i.e., the neutralizingautoantibody will inhibit the activity of both the first and secondbiological drugs.

In certain other embodiments, the absence of the labeled complex is anindication that the neutralizing autoantibody against the first biologicis not cross-reactive with the second biologic, i.e., the neutralizingautoantibody will not inhibit the activity of the second biologicaldrug.

In some embodiments, the first and second biologics are independentlyselected from the group consisting of antibodies (e.g., anti-TNFαmonoclonal antibodies), antibody fragments, proteins (e.g., cytokinessuch as interleukins), polypeptides, peptides, fusion proteins,multivalent binding proteins, antibody-drug conjugates, vaccines,nucleic acids, sugars, recombinant forms thereof, engineered formsthereof, and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving biologic therapy. In preferredembodiments, the sample is serum. In particular embodiments, the subjecthas a disease or disorder such as, e.g., an autoimmune disease (e.g.,rheumatoid arthritis), an inflammatory disease (e.g., inflammatory boweldisease (IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)),or cancer.

In certain embodiments, the sample has or is suspected of having anautoantibody to the biologic. In other embodiments, the biologicautoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In certain aspects, the assay methods of the present invention furthercomprise an acid dissociation step comprising contacting a sample withan acid prior to, during, and/or after contacting the sample with alabeled biologic and a labeled biologic binding moiety.

In certain other aspects, the assay methods of the present inventioncomprise detecting the presence or level of one or more isotypes of aneutralizing and/or non-neutralizing form of an autoantibody to abiologic in a sample.

In one particular aspect, the present invention provides a method fordetecting the presence of a neutralizing and/or non-neutralizing form ofan autoantibody to an anti-TNFα drug in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled anti-TNFα drug and a        labeled TNFα to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug and the            autoantibody (i.e., wherein the components of the first            labeled complex are not covalently attached to each other);            and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug, the labeled TNFα,            and the autoantibody (i.e., wherein the components of the            second labeled complex are not covalently attached to each            other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled TNFα, free labeled        anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and        labeled TNFα;    -   (c) measuring the level of free labeled TNFα after size        exclusion chromatography (e.g., by measuring the area under the        curve (AUC) of the free labeled TNFα peak following size        exclusion chromatography (SEC)); and    -   (d) comparing the level of the free labeled TNFα measured in        step (c) to the level of free labeled TNFα in a control sample        (e.g., by measuring the AUC of the free labeled TNFα peak        following SEC of a reference sample containing only free labeled        TNFα), thereby detecting the presence of a neutralizing and/or        non-neutralizing form of the autoantibody.

In some embodiments, a neutralizing form of the autoantibody interfereswith the binding between the anti-TNFα drug and TNFα. In otherembodiments, a non-neutralizing form of the autoantibody does notinterfere with the binding between the anti-TNFα drug and TNFα.

In some instances, free labeled TNFα consists of labeled TNFα that issubstantially free of bound anti-TNFα drug (e.g., labeled and/orunlabeled anti-TNFα drug).

In certain embodiments, a neutralizing form of the autoantibody isdetected when the level of the free labeled TNFα measured in step (c) isthe same or substantially the same as the level of the free labeled TNFαin the control sample. In certain other embodiments, a non-neutralizingform of the autoantibody is detected when the level of the free labeledTNFα measured in step (c) is decreased (e.g., substantially decreased)or absent (e.g., undetectable) compared to the level of the free labeledTNFα in the control sample.

In particular embodiments, the level of the free labeled TNFα measuredin step (c) is considered to be substantially the same as the level ofthe free labeled TNFα in the control sample when it is at least about70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% the level of the free labeledTNFα measured in the control sample. In particular embodiments, thelevel of the free labeled TNFα measured in step (c) is considered to besubstantially decreased compared to the level of the free labeled TNFαin the control sample when it is at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, or 95% less than the level of the free labeled TNFαmeasured in the control sample.

In certain embodiments, the level of free labeled TNFα is measured byintegrating the area under the curve (AUC) of the free labeled TNFα peakfrom a plot of signal intensity as a function of elution time from thesize exclusion chromatography (e.g., SEC-HPLC).

In some embodiments, the anti-TNFα drug is selected from the groupconsisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™(adalimumab), CIMZIA® (certolizumab pegol), SIMPONI° (golimumab; CNTO148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving anti-TNFα drug therapy. Inpreferred embodiments, the sample is serum. In particular embodiments,the subject has a TNFα-mediated disease or disorder such as, e.g., anautoimmune disease (e.g., rheumatoid arthritis) or an inflammatorydisease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease(CD) or ulcerative colitis (UC)).

In certain embodiments, the sample has or is suspected of having anautoantibody to the anti-TNFα drug. In other embodiments, the anti-TNFαdrug autoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In another particular aspect, the present invention provides a methodfor measuring the level or percent of a neutralizing form of anautoantibody to an anti-TNFα drug in a sample, the method comprising:

-   -   (a) contacting the sample with a labeled anti-TNFα drug and a        labeled TNFα to form:        -   (i) a first labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug and the            autoantibody (i.e., wherein the components of the first            labeled complex are not covalently attached to each other);            and/or        -   (ii) a second labeled complex (i.e., immuno-complex or            conjugate) of the labeled anti-TNFα drug, the labeled TNFα,            and the autoantibody (i.e., wherein the components of the            second labeled complex are not covalently attached to each            other);    -   (b) subjecting the first labeled complex and/or the second        labeled complex to size exclusion chromatography to separate        them from free (i.e., unbound) labeled TNFα, free labeled        anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and        labeled TNFα;    -   (c) measuring the level of free labeled TNFα after size        exclusion chromatography (e.g., by measuring the area under the        curve (AUC) of the free labeled TNFα peak following size        exclusion chromatography (SEC)); and    -   (d) comparing the level of free labeled TNFα measured in        step (c) to a normalized level or percent of free labeled TNFα        in a control sample (e.g., by measuring and normalizing the AUC        of the free labeled TNFα peak following SEC of a reference        sample containing only free labeled TNFα to calculate the level        or percent of free labeled TNFα), wherein the normalized level        or percent of the free labeled TNFα in the control sample        corresponds to the level or percent of a neutralizing form of        the autoantibody.

In some embodiments, the difference between the normalized level orpercent of the free labeled TNFα in the control sample and the level offree labeled TNFα measured in step (c) corresponds to the level orpercent of a non-neutralizing form of the autoantibody.

In some instances, free labeled TNFα consists of labeled TNFα that issubstantially free of bound anti-TNFα drug (e.g., labeled and/orunlabeled anti-TNFα drug).

In particular embodiments, the level or percent of the free labeled TNFαin a control sample is normalized by measuring the peak area (e.g., bymeasuring the AUC) of a complex formed between the labeled anti-TNFαdrug and labeled TNFα (e.g., “labeled complex”), and then subtractingthe measured peak area of the labeled complex from the peak area of thefree labeled TNFα (e.g., by measuring the AUC of the free labeled TNFαpeak).

In certain embodiments, the level of free labeled TNFα is measured byintegrating the area under the curve (AUC) of the free labeled TNFα peakfrom a plot of signal intensity as a function of elution time from thesize exclusion chromatography (e.g., SEC-HPLC). In other embodiments,the level of a complex formed between the labeled anti-TNFα drug andlabeled TNFα is measured by integrating the AUC of the free labeled TNFαpeak from a plot of signal intensity as a function of elution time fromthe size exclusion chromatography (e.g., SEC-HPLC).

In certain embodiments, a subpopulation of the autoantibody to ananti-TNFα drug (e.g., ADA) is a neutralizing form of the autoantibody(e.g., NAb). In some embodiments, the total level of an autoantibody toan anti-TNFα drug in a sample can be calculated by adding the levels ofboth neutralizing and non-neutralizing forms of the autoantibodymeasured in accordance with the methods of the invention.

In some embodiments, the level of the free labeled TNFα measured in step(c) is further compared to a negative control, a positive control, or acombination thereof. Non-limiting examples of negative controls includea mouse monoclonal anti-human IgG₁ Fc sample and/or a rabbit monoclonalanti-human IgG₁ Fc sample. Non-limiting examples of positive controlsinclude a pooled ADA-positive patient serum sample and/or a sample ofrabbit polyclonal antibodies against the F(ab′)₂ fragment of ananti-TNFα drug.

In further embodiments, the percent of the neutralizing form of theautoantibody (e.g., NAb) determined in step (d) is compared to a cutoffvalue or reference range established from a healthy control (e.g.,normal human serum). In particular embodiments, the cutoff value orreference range is expressed as a threshold percent of NAb that thesample must have in order to be considered positive for NAb. In suchembodiments, the sample is positive for NAb when the percent of NAbdetermined in step (d) is greater than or equal to the cutoff value orreference range established from the healthy control. In otherembodiments, the sample is negative for NAb when the percent of NAbdetermined in step (d) is less than the cutoff value or reference rangeestablished from the healthy control. Non-limiting examples of cutoffvalues or reference ranges include, e.g., at least about 0.25%, 0.50%,0.75%, 1.00%, 1.50%, 2.00%, 2.50%, 2.60%, 2.70%, 2.80%, 2.90%, 3.00%,3.01%, 3.02%, 3.03%, 3.04%, 3.05%, 3.06%, 3.07%, 3.08%, 3.09%, 3.10%,3.20%, 3.30%, 3.40%, 3.50%, 4.00%, 4.50%, 5.00%, 5.50%, 6.00%, 6.50%,7.00%, 7.50%, 8.00%, 8.50%, 9.00%, 9.50%, 10.00% NAb, or any rangetherein. In some instances, the cutoff value or reference range is about3.00% NAb or about 3.06% NAb or between about 3.00%-3.10% NAb.

In some embodiments, all the autoantibodies to the anti-TNFα drug areneutralizing antibodies and the sample is defined as having 100%neutralizing anti-drug antibodies (NAb) and/or 0% non-neutralizinganti-drug antibodies (non-NAb). In these embodiments, the level of thefree labeled TNFα measured in step (c) is generally the same as thelevel of the free labeled TNFα in the control sample, and theautoantibodies are predicted to completely block or interfere with thebinding between the anti-TNFα drug and TNFα.

In certain other embodiments, none of the autoantibodies to theanti-TNFα drug are neutralizing antibodies and the sample is defined ashaving 100% non-NAb and/or 0% NAb. In these embodiments, the level ofthe free labeled TNFα measured in step (c) is generally absent (e.g.,undetectable) compared to the level of the free labeled TNFα in thecontrol sample, and the autoantibodies are predicted to not completelyblock or interfere with the binding between the anti-TNFα drug and TNFα.

In further embodiments, when both neutralizing and non-neutralizingforms of the autoantibody are present in a sample, the percent of eachspecies can be expressed on their own (e.g., 50% NAb or 50% non-NAb isdefined as an equal proportion of NAb and non-NAb in a sample) or as aratio. In certain instances, the ratio is calculated by dividing thepercent of NAb by the percent of non-NAb, or vice versa. In otherinstances, the ratio is calculated by dividing the level of NAb by thelevel of non-NAb, or vice versa.

In some embodiments, the anti-TNFα drug is selected from the groupconsisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™(adalimumab), CIMZIA® (certolizumab pegol), SIMPONI° (golimumab; CNTO148), and combinations thereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving anti-TNFα drug therapy. Inpreferred embodiments, the sample is serum. In particular embodiments,the subject has a TNFα-mediated disease or disorder such as, e.g., anautoimmune disease (e.g., rheumatoid arthritis) or an inflammatorydisease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease(CD) or ulcerative colitis (UC)).

In certain embodiments, the sample has or is suspected of having anautoantibody to the anti-TNFα drug. In other embodiments, the anti-TNFαdrug autoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In yet another particular aspect, the present invention provides amethod for determining whether a neutralizing form of an autoantibody toa first anti-TNFα drug is cross-reactive with a second (i.e., different)anti-TNFα drug, the method comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of the autoantibody in a sample in accordance        with an assay described herein to determine whether the sample        is positive or negative for the neutralizing form of the        autoantibody; and

if the sample is positive for the neutralizing form of the autoantibody,then:

-   -   (b) contacting the sample with a labeled second anti-TNFα drug        to form a labeled complex of the labeled second anti-TNFα drug        and the neutralizing form of the autoantibody (i.e., wherein the        components of the labeled complex are not covalently attached to        each other);    -   (c) subjecting the labeled complex to size exclusion        chromatography to separate the labeled complex (e.g., from free        labeled second anti-TNFα drug); and    -   (d) detecting the labeled complex, thereby determining whether a        neutralizing form of an autoantibody to a first anti-TNFα drug        is cross-reactive with a second anti-TNFα drug.

In certain embodiments, the presence of the labeled complex is anindication that the neutralizing autoantibody against the firstanti-TNFα drug is cross-reactive with the second anti-TNFα drug, i.e.,the neutralizing autoantibody will inhibit the activity of both thefirst and second anti-TNFα drugs.

In certain other embodiments, the absence of the labeled complex is anindication that the neutralizing autoantibody against the firstanti-TNFα drug is not cross-reactive with the second anti-TNFα drug,i.e., the neutralizing autoantibody will not inhibit the activity of thesecond anti-TNFα drug.

In particular embodiments, the first and second anti-TNFα drugs areindependently selected from the group consisting of REMICADE™(infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA®(certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinationsthereof.

In other embodiments, the sample is a whole blood, serum, or plasmasample, e.g., from a subject receiving anti-TNFα drug therapy. Inpreferred embodiments, the sample is serum. In particular embodiments,the subject has a TNFα-mediated disease or disorder such as, e.g., anautoimmune disease (e.g., rheumatoid arthritis) or an inflammatorydisease (e.g., inflammatory bowel disease (IBD) such as Crohn's disease(CD) or ulcerative colitis (UC)).

In certain embodiments, the sample has or is suspected of having anautoantibody to the anti-TNFα drug. In other embodiments, the anti-TNFαdrug autoantibody includes, but is not limited to, human anti-chimericantibodies (HACA), human anti-humanized antibodies (HAHA), and humananti-mouse antibodies (HAMA), as well as combinations thereof.

In certain aspects, the assay methods of the present invention furthercomprise an acid dissociation step comprising contacting a sample withan acid prior to, during, and/or after contacting the sample with alabeled anti-TNFα drug and a labeled TNFα.

Methods for detecting anti-drug antibodies using acid dissociation aredescribed herein and in PCT Application No. PCT/US2012/025437, filedFeb. 16, 2012, the disclosure of which is hereby incorporated byreference in its entirety for all purposes.

In certain other aspects, the assay methods of the present inventioncomprise detecting the presence or level of one or more isotypes of aneutralizing and/or non-neutralizing form of an autoantibody to ananti-TNFα drug in a sample. As a non-limiting example, the assays of thepresent invention can be used to determine different neutralizing and/ornon-neutralizing ADA isotypes in samples from ADA-positive patientsreceiving an anti-TNFα drug such as REMICADE™ (infliximab) or HUMIRA™(adalimumab). In certain embodiments, the one or more isotypes comprisesa plurality of at least two, three, four, five, or more isotypes. Inother embodiments, the one or more isotypes is selected from the groupconsisting of IgA, IgD, IgE, IgG, and IgM isotypes, subclasses thereof,and combinations thereof. In certain embodiments, each autoantibodyisotype is characterized, identified, and/or detected by its retentiontime. In other embodiments, each autoantibody isotype is characterized,identified, and/or detected upon a signal that is generated by theproximity binding of detector moieties such as labeled anti-TNFα drugand labeled anti-Ig antibodies specific for different antibody isotypes.In certain instances, the signal comprises a fluorescent signal that canbe detected by fluorescence resonance energy transfer (FRET).

Methods for detecting anti-drug antibody (ADA) isotypes are furtherdescribed in PCT Publication No. WO 2012/054532, the disclosure of whichis hereby incorporated by reference in its entirety for all purposes.

A biologic (e.g., anti-TNFα drug) or biologic binding moiety (e.g.,TNFα) can be labeled with any of a variety of detectable group(s). Inpreferred embodiments, the biologic (e.g., anti-TNFα drug) and thebiologic binding moiety (e.g., TNFα) comprise different labels. Incertain embodiments, a biologic (e.g., anti-TNFα drug) or biologicbinding moiety (e.g., TNFα) is labeled with a fluorophore or afluorescent dye. Non-limiting examples of fluorophores or fluorescentdyes include those listed in the Molecular Probes Catalogue, which isherein incorporated by reference (see, R. Haugland, The Handbook—A Guideto Fluorescent Probes and Labeling Technologies, 10^(th) Edition,Molecular probes, Inc. (2005)). Such exemplary fluorophores orfluorescent dyes include, but are not limited to, Alexa Fluor® dyes suchas Alexa Fluor® 350, Alexa Fluor® 405, Alexa Fluor® 430, Alexa Fluor®488, Alexa Fluor® 514, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor®555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 610, Alexa Fluor®633, Alexa Fluor® 635, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor®680, Alexa Fluor® 700, Alexa Fluor® 750, and/or Alexa Fluor® 790, aswell as other fluorophores including, but not limited to, DansylChloride (DNS-Cl), 5-(iodoacetamida)fluoroscein (5-IAF), fluoroscein5-isothiocyanate (FITC), tetramethylrhodamine 5- (and 6-)isothiocyanate(TRITC), 6-acryloyl-2-dimethylaminonaphthalene (acrylodan),7-nitrobenzo-2-oxa-1,3,-diazol-4-yl chloride (NBD-Cl), ethidium bromide,Lucifer Yellow, 5-carboxyrhodamine 6G hydrochloride, Lissamine rhodamineB sulfonyl chloride, Texas Red™ sulfonyl chloride, BODIPY™,naphthalamine sulfonic acids (e.g., 1-anilinonaphthalene-8-sulfonic acid(ANS), 6-(p-toluidinyl)naphthalene-2-sulfonic acid (TNS), and the like),Anthroyl fatty acid, DPH, Parinaric acid, TMA-DPH, Fluorenyl fatty acid,fluorescein-phosphatidylethanolamine, TexasRed-phosphatidylethanolamine, Pyrenyl-phophatidylcholine,Fluorenyl-phosphotidylcholine, Merocyanine 540,1-(3-sulfonatopropyl)-4-[β-[2[(di-n-butylamino)-6naphthyl]vinyl]pyridinium betaine (Naphtyl Styryl),3,3′dipropylthiadicarbocyanine (diS-C₃-(5)), 4-(p-dipentylaminostyryl)-1-methylpyridinium (di-5-ASP), Cy-3 Iodo Acetamide,Cy-5-N-Hydroxysuccinimide, Cy-7-Isothiocyanate, rhodamine 800, IR-125,Thiazole Orange, Azure B, Nile Blue, A1 Phthalocyanine, Oxaxine 1,4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33342, TOTO, AcridineOrange, Ethidium Homodimer, N(ethoxycarbonylmethyl)-6-methoxyquinolinium(MQAE), Fura-2, Calcium Green, Carboxy SNARF-6, BAPTA, coumarin,phytofluors, Coronene, metal-ligand complexes, IRDye® 700DX, IRDye® 700,IRDye® 800RS, IRDye® 800CW, IRDye® 800, Cy5, Cy5.5, Cy7, DY676, DY680,DY682, DY780, and mixtures thereof. Additional suitable fluorophoresinclude enzyme-cofactors; lanthanide, green fluorescent protein, yellowfluorescent protein, red fluorescent protein, or mutants and derivatesthereof. In one embodiment of the invention, the second member of thespecific binding pair has a detectable group attached thereto.

Typically, the fluorescent group is a fluorophore selected from thecategory of dyes comprising polymethines, pthalocyanines, cyanines,xanthenes, fluorenes, rhodamines, coumarins, fluoresceins and BODIPY™.

In one embodiment, the fluorescent group is a near-infrared (NIR)fluorophore that emits in the range of between about 650 to about 900nm. Use of near infrared fluorescence technology is advantageous inbiological assays as it substantially eliminates or reduces backgroundfrom auto fluorescence of biosubstrates. Another benefit to the near-IRfluorescent technology is that the scattered light from the excitationsource is greatly reduced since the scattering intensity is proportionalto the inverse fourth power of the wavelength. Low backgroundfluorescence and low scattering result in a high signal to noise ratio,which is essential for highly sensitive detection. Furthermore, theoptically transparent window in the near-IR region (650 nm to 900 nm) inbiological tissue makes NIR fluorescence a valuable technology for invivo imaging and subcellular detection applications that require thetransmission of light through biological components. Within aspects ofthis embodiment, the fluorescent group is preferably selected form thegroup consisting of IRDye® 700DX, IRDye® 700, IRDye® 800RS, IRDye®800CW, IRDye® 800, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700,Alexa Fluor® 750, Alexa Fluor® 790, Cy5, Cy5.5, Cy7, DY 676, DY680,DY682, and DY780. In certain embodiments, the near infrared group isIRDye® 800CW, IRDye® 800, IRDye® 700DX, IRDye® 700, or Dynomic DY676.

Fluorescent labeling is accomplished using a chemically reactivederivative of a fluorophore. Common reactive groups include aminereactive isothiocyanate derivatives such as FITC and TRITC (derivativesof fluorescein and rhodamine), amine reactive succinimidyl esters suchas NHS-fluorescein, and sulfhydryl reactive maleimide activated fluorssuch as fluorescein-5-maleimide, many of which are commerciallyavailable. Reaction of any of these reactive dyes with a biologic (e.g.,anti-TNFα drug) or biologic binding moiety (e.g., TNFα) results in astable covalent bond formed between a fluorophore and a biologic (e.g.,anti-TNFα drug) or biologic binding moiety (e.g., TNFα).

In certain instances, following a fluorescent labeling reaction, it isoften necessary to remove any nonreacted fluorophore from the labeledtarget molecule. This is often accomplished by size exclusionchromatography, taking advantage of the size difference betweenfluorophore and labeled protein.

Reactive fluorescent dyes are available from many sources. They can beobtained with different reactive groups for attachment to variousfunctional groups within the target molecule. They are also available inlabeling kits that contain all the components to carry out a labelingreaction. In one preferred aspect, Alexa Fluor® 647 C2 maleimide is usedfrom Invitrogen (Cat. No. A-20347).

Specific immunological binding of a neutralizing and/or non-neutralizinganti-drug antibody (e.g., NAb and/or non-NAb) to a biologic (e.g.,anti-TNFα drug) and/or biologic binding moiety (e.g., TNFα) can bedetected directly or indirectly. Direct labels include fluorescent orluminescent tags, metals, dyes, radionuclides, and the like, attached tothe antibody. In certain instances, a biologic (e.g., anti-TNFα drug) orbiologic binding moiety (e.g., TNFα) labeled with differentradionuclides can be used for determining the presence or level of NAband/or non-NAb in a sample. In other instances, a chemiluminescenceassay using chemiluminescent biologic (e.g., anti-TNFα drug) andbiologic binding moiety (e.g., TNFα) is suitable for sensitive,non-radioactive detection of the presence or level of NAb and/or non-NAbin a sample. In particular instances, a biologic (e.g., anti-TNFα drug)and biologic binding moiety (e.g., TNFα) labeled with differentfluorochromes is suitable for detection of the presence or level of NAband/or non-NAb in a sample. Examples of fluorochromes include, withoutlimitation, Alexa Fluor® dyes, DAPI, fluorescein, Hoechst 33258,R-phycocyanin, B-phycoerythrin, R-phycoerythrin, rhodamine, Texas red,and lissamine Secondary antibodies linked to fluorochromes can beobtained commercially, e.g., goat F(ab′)₂ anti-human IgG-FITC isavailable from Tago Immunologicals (Burlingame, Calif.).

Indirect labels include various enzymes well-known in the art, such ashorseradish peroxidase (HRP), alkaline phosphatase (AP),β-galactosidase, urease, and the like. A horseradish-peroxidasedetection system can be used, for example, with the chromogenicsubstrate tetramethylbenzidine (TMB), which yields a soluble product inthe presence of hydrogen peroxide that is detectable at 450 nm. Analkaline phosphatase detection system can be used with the chromogenicsubstrate p-nitrophenyl phosphate, for example, which yields a solubleproduct readily detectable at 405 nm. Similarly, a β-galactosidasedetection system can be used with the chromogenic substrateo-nitrophenyl-β-D-galactopyranoside (ONPG), which yields a solubleproduct detectable at 410 nm. An urease detection system can be usedwith a substrate such as urea-bromocresol purple (Sigma Immunochemicals;St. Louis, Mo.). A useful secondary antibody linked to an enzyme can beobtained from a number of commercial sources, e.g., goat F(ab′)₂anti-human IgG-alkaline phosphatase can be purchased from JacksonImmunoResearch (West Grove, Pa.).

A signal from the direct or indirect label can be analyzed, for example,using a spectrophotometer to detect color from a chromogenic substrate;a radiation counter to detect radiation such as a gamma counter fordetection of ¹²⁵I; or a fluorometer to detect fluorescence in thepresence of light of a certain wavelength. For detection ofenzyme-linked antibodies, a quantitative analysis of NAb and/or non-NAblevels can be made using a spectrophotometer such as an EMAX MicroplateReader (Molecular Devices; Menlo Park, Calif.) in accordance with themanufacturer's instructions. If desired, the assays of the presentinvention can be automated or performed robotically, and the signal frommultiple samples can be detected simultaneously.

In certain embodiments, size exclusion chromatography is used. Theunderlying principle of SEC is that particles of different sizes willelute (filter) through a stationary phase at different rates. Thisresults in the separation of a solution of particles based on size.Provided that all the particles are loaded simultaneously or nearsimultaneously, particles of the same size elute together. Each sizeexclusion column has a range of molecular weights that can be separated.The exclusion limit defines the molecular weight at the upper end ofthis range and is where molecules are too large to be trapped in thestationary phase. The permeation limit defines the molecular weight atthe lower end of the range of separation and is where molecules of asmall enough size can penetrate into the pores of the stationary phasecompletely and all molecules below this molecular mass are so small thatthey elute as a single band.

In certain aspects, the eluent is collected in constant volumes, orfractions. The more similar the particles are in size, the more likelythey will be in the same fraction and not detected separately.Preferably, the collected fractions are examined by spectroscopictechniques to determine the concentration of the particles eluted.Typically, the spectroscopy detection techniques useful in the presentinvention include, but are not limited to, fluorometry, refractive index(RI), and ultraviolet (UV). In certain instances, the elution volumedecreases roughly linearly with the logarithm of the molecularhydrodynamic volume (i.e., heavier moieties come off first).

In a further aspect, the present invention provides a method formonitoring and/or optimizing therapy to a biologic in a subjectreceiving a course of therapy with the biologic, the method comprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of an autoantibody to the biologic in        accordance with the assay described herein at a plurality of        time points over the course of therapy;    -   (b) detecting a change in the presence, level, or percent of the        neutralizing form of the autoantibody over time; and    -   (c) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the        presence, level, or percent of the neutralizing form of the        autoantibody over time.

In certain embodiments, the plurality of time points comprises at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, or more time points.

In one particular aspect, the present invention provides a method formonitoring and/or optimizing therapy to a biologic in a subjectreceiving a course of therapy with the biologic, the method comprising:

-   -   (a) measuring the level or percent of a neutralizing form of an        autoantibody to the biologic in a first sample from the subject        as described herein at time point t₀;    -   (b) measuring the level or percent of the neutralizing form of        the autoantibody in a second sample from the subject as        described herein at time point t₁;    -   (c) optionally repeating step (b) with n additional samples from        the subject at time points t_(n+1), wherein n is an integer from        1 to about 25 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or any        range therein);    -   (d) detecting a change in the level or percent of the        neutralizing form of the autoantibody from time points t₀ to t₁        or from time points t₀ to t_(n+1); and    -   (e) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the level        or percent of the neutralizing form of the autoantibody over        time.

In certain other embodiments, the level or percent of the neutralizingform of the autoantibody (e.g., NAb) is measured during the course ofbiologic drug therapy at one or more (e.g., a plurality) of thefollowing weeks: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 80, 90, 100, etc.

In some embodiments, determining a subsequent dose of the course oftherapy for the subject comprises maintaining, increasing, or decreasinga subsequent dose of the course of therapy for the subject. In otherembodiments, determining a different course of therapy for the subjectcomprises treatment with a different biologic drug. In otherembodiments, determining a different course of therapy for the subjectcomprises treatment with the current course of therapy along withanother therapeutic agent. In further embodiments, determining adifferent course of therapy for the subject comprises changing thecurrent course of therapy (e.g., switching to a different biologic or toa drug that targets a different mechanism).

In particular embodiments, an increase in the level or percent of theneutralizing form of the autoantibody (e.g., NAb) over time is anindication that treatment adjustment should be recommended for thesubject. In certain other embodiments, a change from an absence of theneutralizing form of the autoantibody (e.g., NAb) to the presencethereof over time is an indication that treatment adjustment should berecommended for the subject. In these embodiments, the subject can betreated with the current course of therapy (e.g., taking the existingbiologic) along with one or more other therapeutic agents. In certainalternative embodiments, the subject can be switched to a differentbiologic. In certain other alternative embodiments, the subject can beswitched to a drug (e.g., biologic and/or non-biologic) that targets adifferent mechanism.

In an additional aspect, the present invention provides a method foroptimizing therapy and/or reducing toxicity in a subject receiving acourse of therapy with a first biologic, the method comprising:

-   -   (a) determining whether a neutralizing form of an autoantibody        to the first biologic is cross-reactive with a second (i.e.,        different) biologic by detecting or measuring the presence,        level, or percent of a neutralizing form of the autoantibody in        a sample from the subject in accordance with an assay described        herein; and    -   (b) determining that a different course of therapy should be        administered to the subject if the neutralizing form of the        autoantibody is cross-reactive with the second biologic.

In certain embodiments, determining that a different course of therapyshould be administered comprises switching to a drug (e.g., biologicand/or non-biologic) that targets a different mechanism.

In some embodiments, the method further comprises determining that asubsequent dose of the current course of therapy be increased ordecreased, or that a different course of therapy should be administeredto the subject if the neutralizing form of the autoantibody is notcross-reactive with the second biologic. In certain instances, thedifferent course of therapy comprises treatment with the secondbiologic. In certain other instances, the different course of therapycomprises treatment with the first or second biologic along with one ormore other therapeutic agents.

In one particular aspect, the present invention provides a method formonitoring and/or optimizing therapy to an anti-TNFα drug in a subjectreceiving a course of therapy with the anti-TNFα drug, the methodcomprising:

-   -   (a) detecting or measuring the presence, level, or percent of a        neutralizing form of an autoantibody to the anti-TNFα drug in        accordance with the assay described herein at a plurality of        time points over the course of therapy;    -   (b) detecting a change in the presence, level, or percent of the        neutralizing form of the autoantibody over time; and    -   (c) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the        presence, level, or percent of the neutralizing form of the        autoantibody over time.

In certain embodiments, the plurality of time points comprises at least2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, or more time points.

In another particular aspect, the present invention provides a methodfor monitoring and/or optimizing therapy to an anti-TNFα drug in asubject receiving a course of therapy with the anti-TNFα drug, themethod comprising:

-   -   (a) measuring the level or percent of a neutralizing form of an        autoantibody to the anti-TNFα drug in a first sample from the        subject as described herein at time point t₀;    -   (b) measuring the level or percent of the neutralizing form of        the autoantibody in a second sample from the subject as        described herein at time point t₁;    -   (c) optionally repeating step (b) with n additional samples from        the subject at time points t_(n+1), wherein n is an integer from        1 to about 25 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,        13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, or any        range therein);    -   (d) detecting a change in the level or percent of the        neutralizing form of the autoantibody from time points t₀ to t₁        or from time points t₀ to t_(n+1); and    -   (e) determining a subsequent dose of the course of therapy for        the subject or whether a different course of therapy should be        administered to the subject based upon the change in the level        or percent of the neutralizing form of the autoantibody over        time.

In certain other embodiments, the level or percent of the neutralizingform of the autoantibody (e.g., NAb) is measured during the course ofanti-TNFα drug therapy at one or more (e.g., a plurality) of thefollowing weeks: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48,50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 80, 90, 100, etc.

In some embodiments, determining a subsequent dose of the course oftherapy for the subject comprises maintaining, increasing, or decreasinga subsequent dose of the course of therapy for the subject. In otherembodiments, determining a different course of therapy for the subjectcomprises treatment with a different anti-TNFα drug. In otherembodiments, determining a different course of therapy for the subjectcomprises treatment with the current course of therapy along withanother therapeutic agent including, but not limited to, an anti-TNFtherapy, an immunosuppressive agent, a corticosteroid, a drug thattargets a different mechanism, a nutrition therapy, and othercombination treatments. In further embodiments, determining a differentcourse of therapy for the subject comprises changing the current courseof therapy (e.g., switching to a different anti-TNF drug or to a drugthat targets a different mechanism such as an IL-6 receptor-inhibitingmonoclonal antibody, anti-integrin molecule (e.g., Tysabri,Vedaluzamab), JAK-2 inhibitor, and tyrosine kinase inhibitor, or to anutrition therapy (e.g., special carbohydrate diet)).

In particular embodiments, an increase in the level or percent of theneutralizing form of the autoantibody (e.g., NAb) over time is anindication that treatment adjustment should be recommended for thesubject. In certain other embodiments, a change from an absence of theneutralizing form of the autoantibody (e.g., NAb) to the presencethereof over time is an indication that treatment adjustment should berecommended for the subject. In these embodiments, the subject can betreated with the current course of therapy (e.g., taking the existinganti-TNFα drug) along with one or more immunosuppressive agents such as,e.g., methotrexate (MTX) or azathioprine (AZA). In certain alternativeembodiments, the subject can be switched to a different anti-TNFα drug.In certain other alternative embodiments, the subject can be switched toa drug that targets a different mechanism (e.g., a non-anti-TNFα drug).

In yet another particular aspect, the present invention provides amethod for optimizing therapy and/or reducing toxicity in a subjectreceiving a course of therapy with a first anti-TNFα drug, the methodcomprising:

-   -   (a) determining whether a neutralizing form of an autoantibody        to the first anti-TNFα drug is cross-reactive with a second        (i.e., different) anti-TNFα drug by detecting or measuring the        presence, level, or percent of a neutralizing form of the        autoantibody in a sample from the subject in accordance with an        assay described herein; and    -   (b) determining that a different course of therapy should be        administered to the subject if the neutralizing form of the        autoantibody is cross-reactive with the second anti-TNFα drug.

In certain embodiments, determining that a different course of therapyshould be administered comprises switching to a drug that targets adifferent mechanism (e.g., a non-anti-TNFα drug). Non-limiting examplesof such drugs include an IL-6 receptor-inhibiting monoclonal antibody,anti-integrin molecule (e.g., Tysabri, Vedaluzamab), JAK-2 inhibitor,tyrosine kinase inhibitor, a nutrition therapy (e.g., specialcarbohydrate diet), and mixtures thereof.

In some embodiments, the method further comprises determining that asubsequent dose of the current course of therapy be increased ordecreased, or that a different course of therapy should be administeredto the subject if the neutralizing form of the autoantibody is notcross-reactive with the second anti-TNFα drug. In certain instances, thedifferent course of therapy comprises treatment with the secondanti-TNFα drug. In certain other instances, the different course oftherapy comprises treatment with the first or second anti-TNFα drugalong with one or more immunosuppressive agents such as MTX or AZA.

Methods for detecting anti-TNFα drugs and anti-drug antibodies arefurther described in PCT Publication No. WO 2011/056590, the disclosureof which is hereby incorporated by reference in its entirety for allpurposes.

In certain instances, the present invention may further compriseadministering to a subject a therapeutically effective amount of acourse of therapy such as an anti-TNFα drug or a drug that targets adifferent mechanism (e.g., a non-anti-TNFα drug) useful for treating oneor more symptoms associated with a TNFα-mediated disease or disorder(e.g., IBD such as CD or UC). For therapeutic applications, the courseof therapy can be administered alone or co-administered in combinationwith one or more additional agents as described herein. As such, thepresent invention advantageously enables a clinician to practice“personalized medicine” by guiding treatment decisions and informingtherapy selection and optimization for anti-TNFα drugs such that theright drug is given to the right patient at the right time.

IV. Acid Dissociation

In certain aspects, the assay methods of the present invention furthercomprise an acid dissociation step, e.g., to enable equilibration ofimmune complexes for measuring the presence or level of neutralizingautoantibodies (NAb), non-neutralizing autoantibodies (non-NAb), and/orisotypes thereof that are generated against biologics such as anti-TNFαdrugs. As a result, the presence or level of NAb and/or non-NAb to abiologic (e.g., anti-TNFα drug) administered to a subject in needthereof can be measured without substantial interference from theadministered biologic that is also present in the subject's sample. Inparticular, a subject's sample can be incubated with an amount of acidthat is sufficient to provide for the measurement of the presence orlevel of NAb and/or non-NAb in the presence of the biologic (e.g.,anti-TNFα drug) but without substantial interference from high biologicdrug levels.

In some embodiments, step (a) of the assay methods of the presentinvention may comprise:

-   -   (a′) contacting the sample with an acid to dissociate preformed        complexes of the autoantibody (e.g., including neutralizing        and/or non-neutralizing forms thereof) and the biologic (e.g.,        anti-TNFα drug);    -   (b′) contacting the sample with a labeled biologic (e.g.,        anti-TNFα drug) and a labeled biologic binding moiety (e.g.,        TNFα) following dissociation of the preformed complexes; and    -   (c′) neutralizing the acid in the sample to form:        -   (i) a first labeled complex of the labeled biologic (e.g.,            anti-TNFα drug) and the autoantibody; and/or        -   (ii) a second labeled complex of the labeled biologic (e.g.,            anti-TNFα drug), the labeled biologic binding moiety (e.g.,            TNFα), and the autoantibody.

In some alternative embodiments, steps (a′) and (b′) are performedsimultaneously, e.g., the sample is contacted with an acid, a labeledbiologic (e.g., anti-TNFα drug), and a labeled biologic binding moiety(e.g., TNFα) at the same time. In other alternative embodiments, step(b′) is performed prior to step (a′), e.g., the sample is firstcontacted with a labeled biologic (e.g., anti-TNFα drug) and a labeledbiologic binding moiety (e.g., TNFα), and then contacted with an acid.In further embodiments, steps (b′) and (c′) are performedsimultaneously, e.g., the sample is contacted with a labeled biologic(e.g., anti-TNFα drug) and a labeled biologic binding moiety (e.g.,TNFα) and neutralized (e.g., by contacting the sample with one or moreneutralizing agents) at the same time.

In particular embodiments, the sample is contacted with an amount of anacid that is sufficient to dissociate preformed complexes of theautoantibody and the biologic (e.g., anti-TNFα drug), such that thelabeled biologic binding moiety (e.g., TNFα), the labeled biologic(e.g., anti-TNFα drug), the unlabeled biologic (e.g., anti-TNFα drug),and the autoantibody to the biologic (e.g., anti-TNFα drug) canequilibrate and form complexes therebetween. In certain embodiments, thesample can be contacted with an amount of an acid that is sufficient toallow for the detection and/or measurement of the autoantibody in thepresence of a high level of the biologic (e.g., anti-TNFα drug).

In some embodiments, the phrase “high level of a biologic” such as ahigh level of an anti-TNFα drug includes drug levels of from about 10 toabout 100 μg/mL, about 20 to about 80 μg/mL, about 30 to about 70 μg/mL,or about 40 to about 80 μg/mL. In other embodiments, the phrase “highlevel of a biologic” such as a high level of an anti-TNFα drug includesdrug levels greater than or equal to about 10, 20, 30, 40, 50, 60, 70,80, 90, or 100 μg/mL.

In some embodiments, the acid comprises an organic acid. In otherembodiments, the acid comprises an inorganic acid. In furtherembodiments, the acid comprises a mixture of an organic acid and aninorganic acid. Non-limiting examples of organic acids include citricacid, isocitric acid, glutamic acid, acetic acid, lactic acid, formicacid, oxalic acid, uric acid, trifluoroacetic acid, benzene sulfonicacid, aminomethanesulfonic acid, camphor-10-sulfonic acid, chloroaceticacid, bromoacetic acid, iodoacetic acid, propanoic acid, butanoic acid,glyceric acid, succinic acid, malic acid, aspartic acid, andcombinations thereof. Non-limiting examples of inorganic acids includehydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, boricacid, hydrofluoric acid, hydrobromic acid, and combinations thereof.

In certain embodiments, the amount of an acid corresponds to aconcentration of from about 0.01M to about 10M, about 0.1M to about 5M,about 0.1M to about 2M, about 0.2M to about 1M, or about 0.25M to about0.75M of an acid or a mixture of acids. In other embodiments, the amountof an acid corresponds to a concentration of greater than or equal toabout 00.1M, 0.05M, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M,0.9M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, or 10M of an acid or a mixtureof acids. The pH of the acid can be, for example, about 0.1, 0.5, 1.0,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, or 6.5.

In some embodiments, the sample is contacted with an acid an amount oftime that is sufficient to dissociate preformed complexes of theautoantibody and the biologic (e.g., anti-TNFα drug). In certaininstances, the sample is contacted (e.g., incubated) with an acid for aperiod of time ranging from about 0.1 hours to about 24 hours, about 0.2hours to about 16 hours, about 0.5 hours to about 10 hours, about 0.5hours to about 5 hours, or about 0.5 hours to about 2 hours. In otherinstances, the sample is contacted (e.g., incubated) with an acid for aperiod of time that is greater than or equal to about 0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7,8, 9, or 10 hours. The sample can be contacted with an acid at 4° C.,room temperature (RT), or 37° C.

In certain embodiments, the step of neutralizing the acid comprisesraising the pH of the sample to allow the formation of first and/orsecond labeled complexes described herein. In some embodiments, the acidis neutralized by the addition of one or more neutralizing agents suchas, for example, strong bases, weak bases, buffer solutions, andcombinations thereof. One skilled in the art will appreciate thatneutralization reactions do not necessarily imply a resultant pH of 7.In some instances, acid neutralization results in a sample that isbasic. In other instances, acid neutralization results in a sample thatis acidic (but higher than the pH of the sample prior to adding theneutralizing agent). In particular embodiments, the neutralizing agentcomprises a buffer such as phosphate buffered saline (e.g., 10×PBS) at apH of about 7.3.

In some embodiments, step (b′) further comprises contacting an internalcontrol with the sample together with a labeled biologic (e.g.,anti-TNFα drug) and a labeled biologic binding moiety (e.g., TNFα)(e.g., before, during, or after dissociation of the preformedcomplexes). In certain instances, the internal control comprises alabeled internal control such as, e.g., Biocytin-Alexa 488. In certainother instances, the amount of the labeled internal control ranges fromabout 1 ng to about 25 ng, about 5 ng to about 25 ng, about 5 ng toabout 20 ng, about 1 ng to about 20 ng, about 1 ng to about 10 ng, orabout 1 ng to about 5 ng per 100 μL of sample analyzed. In furtherinstances, the amount of the labeled internal control is greater than orequal to about 1 ng, 5 ng, 10 ng, 15 ng, 20 ng, or 25 ng per 100 μL ofsample analyzed.

As one non-limiting example of the methods of the present invention,samples such as serum samples (e.g., serum from subjects receivingtherapy with an anti-TNFα drug such as Remicade (IFX)) can be incubatedwith 0.5M citric acid, pH 3.0 for one hour at room temperature.Following the dissociation of preformed complexes between (unlabeled)anti-TNFα drug and autoantibodies to the anti-TNFα drug (e.g., anti-drugantibodies such as anti-IFX antibodies (ATI)), labeled anti-TNFα drug(e.g., IFX-Alexa 488), labeled TNFα (e.g., TNFα-Alexa 532), andoptionally an internal control can be added and the reaction mixture(e.g., immediately) neutralized with a neutralizing agent such as10×PBS, pH 7.3. After neutralization, the reaction mixture can beincubated for another hour at room temperature (e.g., on a plate shaker)to allow equilibration and to complete the reformation of immunecomplexes between the labeled TNFα, the labeled anti-TNFα drug, theunlabeled anti-TNFα drug, and/or the autoantibody to the anti-TNFα drug.The samples can then be filtered and analyzed by SEC-HPLC as describedherein.

In particular embodiments, the methods of the present invention (e.g.,comprising acid dissociation followed by homogeneous solution phasebinding kinetics) significantly increases the IFX drug tolerance suchthat NAb and/or non-NAb ATI can be measured in the presence of IFX up toabout 60 μg/mL. In other words, the methods of the present invention candetect the presence or level of NAb and/or non-NAb to anti-TNFα drugssuch as ATI as well as autoantibodies to other anti-TNFα drugs in thepresence of high levels of anti-TNFα drugs (e.g., IFX), but withoutsubstantial interference therefrom.

Methods for detecting anti-drug antibodies using acid dissociation arefurther described in PCT Application No. PCT/US2012/025437, filed Feb.16, 2012, the disclosure of which is hereby incorporated by reference inits entirety for all purposes.

V. Biologic Therapy

The assays of the present invention are suitable for detecting and/ormeasuring the presence or absence (e.g., whether positive or negative),level, or percent of neutralizing and/or non-neutralizing autoantibodiesto any biologic in a sample from a subject (e.g., a subject receivingbiologic therapy). Non-limiting examples of biologics includeantibodies, antibody fragments, proteins, polypeptides, peptides, fusionproteins (e.g., Ig fusion proteins or Fc fusion proteins), multivalentbinding proteins (e.g., DVD Ig), antibody-drug conjugates, vaccines,nucleic acids, sugars, recombinant forms thereof, engineered formsthereof, and combinations thereof.

Examples of antibody-based biologics include, but are not limited to,therapeutic monoclonal antibodies and antigen-binding fragments thereof.In particular embodiments, the antibody comprises an anti-TNFα drug suchas REMICADE™ (infliximab), HUMIRA™ (adalimumab), CIMZIA® (certolizumabpegol), SIMPONI® (golimumab; CNTO 148), or combinations thereof.Additional examples of antibody-based biologics include antibody-drugconjugates such as Adcetris™ (brentuximab vedotin). Table 1 provides anexemplary list of therapeutic monoclonal antibodies which have eitherbeen approved or are currently in development. An extensive list ofmonoclonal antibody therapeutics in clinical development and approvedproducts is provided in the 2006 PhRMA Report entitled “418Biotechnology Medicines in Testing Promise to Bolster the ArsenalAgainst Disease,” the disclosure of which is hereby incorporated byreference in its entirety for all purposes.

TABLE 1 Therapeutic monoclonal antibodies Product Name CompanyIndication(s) Inflammatory Diseases Remicade ™ (infliximab) JanssenBiotech, Inc. Crohn's disease ABT 874 Abbott Laboratories Crohn'sdisease Stelara ® (ustekinumab) Janssen Biotech, Inc. Crohn's diseaseHumira ™ (adalimumab) Abbott Laboratories Crohn's disease MDX-1100Millennium Pharmaceuticals ulcerative colitis Nuvion ® (visilizumab) PDLBioPharma I.V. steroid-refractory ulcerative colitis and Crohn's diseaseTysarbi ® (natalizumab) Biogen Idec Crohn's disease Simponi ®(golimumab) Janssen Biotech, Inc. uveitis Autoimmune disorders Humira ™(adalimumab) Abbott Laboratories rheumatoid arthritis, ankylosingspondylitis, juvenile rheumatoid arthritis, psoriasis Remicade ™(infliximab) Janssen Biotech, Inc. rheumatoid arthritis Simponi ®(golimumab) Janssen Biotech, Inc. rheumatoid arthritis, ankylosingspondylitis, psoriatic arthritis Rituxan ® (rituximab) Genentechrheumatoid arthritis, lupus, primary Biogen Idec progressive multiplesclerosis, SLE, relapsing-remitting multiple sclerosis Tysarbi ®(natalizumab) Biogen Idec multiple scleorisis Stelara ® (ustekinumab)Janssen Biotech, Inc. plaque psoriasis, multiple sclerosis ART 874Abbott Laboratories multiple sclerosis Actemra Roche rheumatoidarthritis AME 527 Applied Molecular rheumatoid arthritis AMG 108 Amgenrheumatoid arthritis AMG 714 Amgen rheumatoid arthritis anti-CD16 MAbMacroGenics immune thrombocytopenic daclizumab (anti-CD25 MAb) PDLBioPharma multiple sclerosis Biogen Idec denosumab (AMG 162) Amgenrheumatoid arthritis ETI-201 Elusys Therapeutics SLE HuMax-CD20(ofatumumab) Genmab rheumatoid arthritis HuZAF ™ (fontolizumab) PDLBioPharma rheumatoid arthritis Biogen Idec IMMU-106 (hCD20) Immunomedicsautoimmune disease LymphoStat-B ™ (belimumab) Human Genome Sciencesrheumatoid arthritis, SLE MEDI-545 (MDX-1103) Medarex lupus MedImmunesiplizumab (MEDI-507) MedImmune psoriasis MLN 1202 MillenniumPharmaceuticals multiple sclerosis ocrelizumab (anti-CD20) (R1594)Genentech multiple sclerosis, rheumatoid Biogen Idec arthritis RocheOKT3-gamma-1 Johnson & Johnson psoriatic arthritis TRX 1 (anti-CD4)TolerRx cutaneous lupus erythematosus TRX 4 TolerRx psoriasis Infectiousdiseases Synagis ® (palivizumab) MedImmune prevention of respiratorysyncytial virus (RSV) MDX-066 (CDA-1) Medarex C. difficile diseaseanti-HIV-1 MAb Polymun Scientific HIV infection CCR5 MAb Hunan GenomeSciences HIV infection Cytolin ® (anti-CD8 MAb) CytoDyn HIV infectionNM01 SRD Pharmaceuticals HIV infection PRO 140 Progenics PharmaceuticalsHIV infection TNX-355 Tanox HIV infection ABthrax ™ (raxibcumab) HumanGenome Sciences anthrax Anthim ™(ETI-204) Elusys Therapeutics anthraxanti-hsp90 MAb NeuTec Pharma candidiasis anti-staph MAb MedImmuneprevention of staphylococcal infections Aurexis (tefibazumab) Inhibitexprevention and treatment of S. aureus bacteremia bavituximab PeregrinePharmaceuticals hepatitis C MDX-1303 Medarex anthrax PharmAthene Numax ™(motavizumab) MedImmune RSV Tarvacin ™ Peregrine Pharmaceuticalshepatitis C XTL 6865 XTL Biopharmaceuticals hepatitis C Cancer Avastin ™(bevacizumab) Genentech metastatic colorectal cancer Bexxar ®(tositumomab) GlaxoSmithKline non-Hodgkin's lymphoma Campath ®(alemtuzumab) Berlex Laboratories B-cell chronic lymphocytic Genzymeleukemia Erbitux ™ (cetuximab) Bristol-Myers Squibb colorectal cancer,squamous cell Medarex cancer of the head and neck Herceptin ®(trastuzumab) Genentech HER2-overexpressing early stage or metastaticbreast cancer Mylotarg ™ (gemtuzumab Wyeth acute myeloid leukemiaozogamicin) Rituxan ® (rituximab) Genentech B-cell non-Hodgkin'slymphoma, Biogen Idec indolent non-Hodgkin's lymphoma induction therapy,relapsed or refractory CLL Zevalin ™ (ibritumomab tiuxetan) Biogen IdecNon-Hodgkin's lymphoma 1311-huA33 Life Science Pharmaceuticalscolorectal cancer 1D09C3 GPC Biotech relapsed/refractory B-celllymphomas AGS PSCA MAb Agensys prostate cancer Merck AMG 102 Amgencancer AMG 479 Amgen cancer AMG 623 Amgen B-cell chronic lymphocyticleukemia (CLL) AMG 655 Amgen cancer AMG 706 Amgen imatinib-resistantGIST, advanced thyroid cancer AMG 706 Amgen imatinib resistant GIST,advanced thyroid cancer anti-CD23 MAb Biogen Idec CLL anti-CD80 MAbBiogen Idec non-Hodgkin's B-cell lymphoma anti-idiotype cancer vaccineViventia Biotech malignant melanoma anti-lymphotoxin beta receptorBiogen Idec solid tumors MAb anti-PEM MAb Somanta Pharmaceuticals canceranti-Tac(Fv)-E38 immunotoxin National Cancer Institute leukemia,lymphoma Avastin ® (bevacizumab) Genentech relapsed metastaticcolorectal cancer, first line metastatic breast cancer, first-linenon-squamous NSCLC cancers AVE 9633 maytansin-loaded anti- SanofiAventis AML CD33 MAb bavituximab Peregrine Pharmaceuticals solid cancersCAT 3888 Cambridge Antibody Technology hairy cell leukemia chimeric MAbNational Cancer Institute neuroblastoma siltuximab (CNTO 328) JanssenBiotech, Inc. renal cancer, prostate cancer, multiple myeloma Cotara ™Peregrine Pharmaceuticals brain cancer bivatuzumab Boehringer Ingelheimcancer Pharmaceuticals CP-751871 (figitumumab) Pfizer adrenocorticalcarcinoma, non- small cell lung cancer CS-1008 (tigatuzumab) DaiichiSankyo pancreatic cancer, colorectal cancer, non-small cell lung cancer,ovarian cancer BrevaRex ™ ViRexx breast cancer, multiple myelomadenosumab Amgen bone loss induced by hormone ablation therapy for breastor prostate cancer, prolonging bone metastases-free survival, bonemetastases in breast cancer ecromeximab Kyowa Hakko USA malignantmelanoma EMD 273063 EMD Lexigen solid tumors, malignant melanoma,neuroblastoma, SCLC Erbitux ™ Bristol Myers Squibb head/neck cancer,first-line palicreatic, first-line NSCLC, second-line NSCLC, first linecolorectal cancer, second-line colorectal cancer GMK ProgeniesPharmaceuticals prevention of recurrence following surgery to removeprimacy melanoma in high-risk patients Campath ® (alemtuzumab) NationalCancer Institute leukemia, lymphoma Berlex Laboratories HGS-ETR1 HumanGenome Sciences hematologic and solid tumors HGS ETR2 (mapatumumab)Human Genome Sciences hematologic and solid tumors HGS-TR2J Human GenomeSciences advanced solid tumors HuC242-DM4 ImmunoGen colorectal,gastrointestinal, NSCLC, pancreatic cancers HuMax-CD4 (zanolimumab)Genmab cutaneous T-cell lymphoma, non- Serono cutaneous T-cell lymphomaHuMax CD20 (ofatumumab) Gemnab CLL, non-Hodgkin's lymphoma HuMax-EGFrGenmab head and neck cancer huN901-DM1 ImmunoGen SCLC multiple myelomaipilimumab Bristol-Myers Squibb melanoma monotherapy, leukemia, Medarexlymphoma, ovarian, prostate, renal cell cancers, melanoma (MCX-010 +/−DTIC), second-line metastatic melanoma (MDX-010 disomotide/ overmotideMDX-1379) M195-bismuth 213 conjugate Actinium Pharmaceuticals AML M200(volociximab) PDL BioPharma Fremont, CA advanced solid tumors BiogenIdec Cambridge, MA MAb HeFi-1 National Cancer Institute lymphoma,non-Hodgkin's Bethesda, MD lymphoma MDX-060 (iratumumab) MedarexHodgkin's disease, anaplastic large- cell-lymphoma MDX-070 Medarexprostate cancer MDX-214 Medarex ECFR-expressing cancers MEDI-522MedImmune T-cell lymphoma, melanoma, prostate cancer, solid tumors MORAb003 Morphotek ovarian cancer MORAb 009 Morphotek mesothelin-expressingtumors neuradiab Bradmer Pharmaceuticals glioblastoma nimotuzumab YMBiosciences squamous cell carcinomas of the head and neck, recurrent orrefractory high grade malignant glioma, anaplastic astrocytomas,glioblastomas and diffuse intrinsic pontine glioma Omnitarg ™(pertuzumab) Genentech ovarian cancer OvaRex ® (oregovomab) ViRexx MAbovarian cancer PAM 4 Merck pancreatic cancer panitumumab (rIIuMAb EGFr)Abgenix colorectal cancer PSMA-ADC Progenics Pharmaceuticals prostatecancer R1550 RadioTheraCIM Roche metastatic breast cancer, glioma YMBioSciences RAV 12 Raven Biotechnologies cancer Rencarex ® G250 Wilex AGrenal cancer SGN30 Seattle Genetics cutaneous anaplastic large-cell MAblyrphoma, systemic anaplastic large-cell lymphoma, Hodgkin's diseaseSGN-33 (lintuzumab) Seattle Genetics AML, myelodysplastic syndromes CLLmultiple myeloma, non Hodgkin's lymphoma SGN-40 Seattle Genetics AML,myelodysplastic syndromes CLL multiple myeloma, non Hodgkin's lymphomasibroturtumab Life Science Pharmaceuticals colorectal, head and neck,lung cancers Tarvacin ™ (bavituximab) Peregrine Pharmaceuticals solidtumors tremelimumab Pfizer metastatic melanoma, prostate cancer TNX-650Tanox refractory Hodgkin's lymphoma Zevalin ™ (ibritumomab tiuxetan)Spectrum Pharmaceuticals non-Hodgkin's lymphoma Blood disorders ReoPro ®(abciximab) Eli Lilly adjunct to percutaneous coronary intervention forthe prevention of cardiac ischemic complications urtoxazumab TeijinPharma hemolytic uremic afelimomab Abbot Laboratories sepsis, septicshock eculizumab Alexion Pharmaceuticals paroxysmal nocturnalhemoglobinurea Cardiovascular disease MLN 1202 MillenniumPharmaceuticals atherosclerosis pexelizumab Alexion Pharmaceuticalsacute myocardial infarction, Procter & Gamble Pharmaceuticalscardiopulmonary bypass Diabetes and Related Conditions anti-CD3 MAbMacroGenics type-1 diabetes mellitus OKT3-gamma-1 Johnson & Johnsontype-1 diabetes mellitus TRX 4 (anti-CD3) TolerRx type-1 diabetesmellitus Genetic Disorders Soliris ™ (eculizumab) AlexionPharmaceuticals paroxysmal nocturnal hemoglobinuria (PNH) NeurologicalDisorders RN624 Rinat Neuroscience osteoarthritis pain RN1219 RinatNeuroscience Alzheimer's disease Respiratory Disorders ABN 912 NovartisPharmaceuticals asthma, chronic obstructive pulmonary disorders (COPD)ABX-IL8 Amgen COPD AMG 317 Amgen asthma daclizumab (anti-CD25 MAb)Protein Design Labs asthma Roche MEDI-528 (anti-TL-9 MAb) MedImmuneasthma mepolizumab (anti-TL5 MAb) Glaxo SmithKline asthma and nasalpolyposis TNX-832 Tanox respiratory diseases Houston, TX Xolair ®(omalizumab) Genentech pediatric asthma Novartis PharmaceuticalsTransplatation ORTHOCLONE OKT ® 3 Ortho Biotech acute kidney transplantrejection, (muromomab-CD3) reversal of heart and liver transplantrejection Simulect ® (basiliximab) Novartis Pharmaceuticals preventionof renal transplant rejection Zenapax ® (daclizumab) Roche prophylaxisof acute kidney transplant rejection OKT3-gamma-1 Protein Design Labsrenal transplant rejection Johnson & Johnson Other CR 0002 CuraGenkidney inflammation denosumab (AMG 162) Amgen postmenopausalosteoporosis mepolizumab (anti-IL5 MAb) GlaxoSmithKlinehypereosinophilic syndrome, eosinophlic esophagitis Xolair ®(omalizumab) Genentech peanut allergy Tanox

Non-limiting examples of protein-based or polypeptide-based biologicsinclude cytokines (e.g., interleukins), chemokines, growth factors,blood-production stimulating proteins (e.g., erythropoietin), hormones(e.g., Elonva® (follicle stimulating hormone), growth hormone), enzymes(e.g., Pulmozyme® (dornase alfa)), clotting factors, insulin, albumin,fragments thereof, conservatively modified variants thereof, analogsthereof, and combinations thereof.

Examples of cytokines include, but are not limited to, TNFα, TNF-relatedweak inducer of apoptosis (TWEAK), osteoprotegerin (OPG), IFN-α, IFN-β,IFN-γ, interleukins (e.g., IL-1α, IL-1β, IL-1 receptor antagonist(IL-1ra), IL-2, IL-4, IL-5, IL-6, soluble IL-6 receptor (sIL-6R), IL-7,IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-23, and IL-27),adipocytokines (e.g., leptin, adiponectin, resistin, active or totalplasminogen activator inhibitor-1 (PAI-1), visfatin, and retinol bindingprotein 4 (RBP4)), and combinations thereof. In particular embodiments,the interleukin comprises IL-2 such as Proleukin® (aldesleukin;recombinant IL-2).

Examples of chemokines include, but are not limited to, CXCL1/GRO1/GROα,CXCL2/GRO2, CXCL3/GRO3, CXCL4/PF-4, CXCL5/ENA-78, CXCL6/GCP-2,CXCL7/NAP-2, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1,CXCL13/BCA-1, CXCL14/BRAK, CXCL15, CXCL16, CXCL17/DMC, CCL1, CCL2/MCP-1,CCL3/MIP-1α, CCL4/MIP-1β, CCL5/RANTES, CCL6/C10, CCL7/MCP-3, CCL8/MCP-2,CCL9/CCL10, CCL11/Eotaxin, CCL12/MCP-5, CCL13/MCP-4, CCL14/HCC-1,CCL15/MIP-5, CCL16/LEC, CCL17/TARC, CCL18/MIP-4, CCL19/MIP-3β,CCL20/MIP-3α, CCL21/SLC, CCL22/MDC, CCL23/MPIF1, CCL24/Eotaxin-2,CCL25/TECK, CCL26/Eotaxin-3, CCL27/CTACK, CCL28/MEC, CL1, CL2, CX₃CL1,and combinations thereof.

Non-limiting examples of growth factors include epidermal growth factor(EGF), heparin-binding epidermal growth factor (HB-EGF), vascularendothelial growth factor (VEGF), pigment epithelium-derived factor(PEDF; also known as SERPINF1), amphiregulin (AREG; also known asschwannoma-derived growth factor (SDGF)), basic fibroblast growth factor(bFGF), hepatocyte growth factor (HGF), transforming growth factor-α(TGF-α), transforming growth factor-β (TGF-β1, TGF-β2, TGF-β3, etc.),endothelin-1 (ET-1), keratinocyte growth factor (KGF; also known asFGF7), bone morphogenetic proteins (e.g., BMP1-BMP15), platelet-derivedgrowth factor (PDGF), nerve growth factor (NGF), β-nerve growth factor(β-NGF), neurotrophic factors (e.g., brain-derived neurotrophic factor(BDNF), neurotrophin 3 (NT3), neurotrophin 4 (NT4), etc.), growthdifferentiation factor-9 (GDF-9), granulocyte-colony stimulating factor(G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF),myostatin (GDF-8), erythropoietin (EPO), thrombopoietin (TPO), andcombinations thereof.

Examples of receptor construct-based or fusion protein-based biologicsinclude, but are not limited to, naturally-occurring receptors linked toan immunoglobulin frame (e.g., Orencia® (abatacept; immunoglobin CTLA-4fusion protein), Amevive® (alefacept; IgG1 fusion protein), ENBREL™(etanercept; recombinant human TNF-receptor fusion protein), engineeredproteins combining two different polypeptide species (e.g., Ontak®(denileukin diftitox; engineered protein comprising interleukin-2 anddiphtheria toxin), and combinations thereof.

The present invention can therefore be used in methods for detecting andmeasuring the presence or level of neutralizing and non-neutralizingautoantibodies to biologics such as anti-TNFα drug therapeutics in asample from a subject receiving biologic therapy for one or more of thediseases or disorders referred to herein and Table 1, including one ormore of the following:

Inflammatory diseases, such as inflammatory bowel disease (IBD) (e.g.,Crohn's disease (CD) and ulcerative colitis (UC)), uveitis, sarcoidosis,Wegener's granulomatosis, and other diseases with inflammation as acentral feature;

Autoimmune diseases, such as rheumatoid arthritis (RA), multiplescleorisis (MS), systemic lupus erythematosus (SLE), ankylosingspondylitis (Bechterew's disease), lupus, psoriatic arthritis, juvenileidiopathic arthritis, psoriasis, and erythematosus;

Cancer, such as digestive and gastrointestinal cancers (e.g., colorectalcancer, small intestine (small bowel) cancer; gastrointestinal stromaltumors, gastrointestinal carcinoid tumors, colon cancer, rectal cancer,anal cancer, bile duct cancer, gastric (stomach) cancer; esophagealcancer; appendix cancer; and the like); gallbladder cancer; livercancer; pancreatic cancer; breast cancer; lung cancer (e.g., non-smallcell lung cancer); prostate cancer; ovarian cancer; renal cancer (e.g.,renal cell carcinoma); cancer of the central nervous system; skincancer; choriocarcinomas; head and neck cancers; hematologicalmalignancies (e.g., leukemia, lymphoma such as B-cell non-Hodgkin'slymphoma); osteogenic sarcomas (e.g., Ewing sarcoma); soft tissuesarcomas (e.g., Dermatofibrosarcoma Protuberans (DFSP),rhabdomyosarcoma); other soft tissue malignancies, and papillary thyroidcarcinomas;

Infectious diseases, such as C. difficile disease, respiratory syncytialvirus (RSV), HIV, anthrax, candidiasis, staphylococcal infections, andhepatitis C;

Blood disorders, such as sepsis, septic shock, paroxysmal nocturnalhemoglobinuria, and hemolytic uremic syndrome;

Cardiovascular disease, such as atherosclerosis, acute myocardialinfarction, cardiopulmonary bypass, and angina;

Metabolic disorders, such as diabetes, e.g., type-I diabetes mellitus;

Genetic disorders, such as paroxysmal nocturnal hemoglobinuria (PNH);

Neurological disorders, such as osteoarthritis pain and Alzheimer'sdisease;

Respiratory disorders, such as asthma, chronic obstructive pulmonarydisorders (COPD), nasal polyposis, and pediatric asthma;

Skin diseases, such as psoriasis, including chronic moderate to severeplaque psoriasis;

Transplant rejection, such as acute kidney transplant rejection,reversal of heart and liver transplant rejection, prevention of renaltransplant rejection, prophylaxis of acute kidney transplant rejection,and renal transplant rejection; and/or

Other disorders, such as kidney inflammation, postmenopausalosteoporosis (bone disorders), hypereosinophilic syndrome, eosinophilicesophagitis and peanut allergy.

In particular embodiments, the subject has a TNFα-mediated disease ordisorder such as, e.g., an autoimmune disease (e.g., rheumatoidarthritis) or an inflammatory disease (e.g., inflammatory bowel disease(IBD) such as Crohn's disease (CD) or ulcerative colitis (UC)).

VI. Examples

The present invention will be described in greater detail by way ofspecific examples. The following examples are offered for illustrativepurposes, and are not intended to limit the invention in any manner.Those of skill in the art will readily recognize a variety ofnoncritical parameters which can be changed or modified to yieldessentially the same results.

The examples from PCT Application No. PCT/US2012/025437, filed Feb. 16,2012, are hereby incorporated by reference in their entirety for allpurposes.

Example 1 Development of a Novel Assay to Monitor Neutralizing Anti-DrugAntibody Formation in IBD Patients

This example illustrates a novel homogeneous assay for detecting ormeasuring the presence or level of neutralizing and/or non-neutralizinganti-drug autoantibodies (ADA) in a patient sample (e.g., serum) usingsize exclusion chromatography in the presence of labeled (e.g.,fluorescently labeled) anti-TNFα drug and labeled TNFα. In particularembodiments, this assay is advantageous because it obviates the need forwash steps which remove low affinity ADA, uses distinct labels (e.g.,fluorophores) that allow for detection on the visible and/or IR spectrawhich decreases background and serum interference issues, increases theability to detect neutralizing and/or non-neutralizing ADA in patientswith a low titer due to the high sensitivity of fluorescent labeldetection, and occurs as a liquid phase reaction, thereby reducing thechance of any changes in the epitope by attachment to a solid surfacesuch as an ELISA plate.

Infliximab (IFX) and adalimumab (ADL) are anti-TNF monoclonal antibodiesprescribed for the treatment of inflammatory bowel disease (IBD).Anti-drug antibodies (ADA) often develop during the course of therapy. Aproportion of these ADA are neutralizing antibodies (NAb). While ADAwill negatively impact drug pharmacokinetics, the presence of NAb willadditionally cause loss of drug efficacy through blockage of the drug'sbinding site. This example describes an assay to monitor the developmentof NAb in IBD patients receiving IFX treatment based on a homogenousmobility shift assay (HMSA) platform and shows the correlation betweenantibody-to-infliximab (ATI) maturation and NAb formation.

Methods:

Serum concentrations of IFX and ATI were measured by HMSA as describedin, e.g., PCT Application No. PCT/US2012/025437, filed Feb. 16, 2012,and PCT Publication No. WO 2011/056590, the disclosures of which arehereby incorporated by reference in their entirety for all purposes. Forthe NAb assay, patient serum containing ATI was first acid dissociated,then two labeled proteins (e.g., IFX-Alexa488 and TNF alpha-Alexa532)were added, followed by neutralization. The solution was diluted to 2%serum, injected by HPLC on a size exclusion column and complexesmonitored by fluorescence. The area under the curve (AUC) of the freeTNF-Alexa532 peak in each spectrum (e.g., plot or chromatogram) wascalculated for controls and patient samples and then a percent NAbcalculated. ATI that completely block antigen binding are defined as100% NAb, 50% means that an equal proportion of ATI in the sample isnon-NAb, and 0% means that all ATI is non-NAb. A reference range wasestablished using serum from 75 healthy volunteers. ATI positive serumsamples (>3.13 U/mL) from 132 residual IBD patient serum screened forIFX and ATI levels were analyzed for NAb. Positive controls were createdusing pooled ATI positive patient serum.

For data analysis, a peak detection algorithm is used to find all of thepeaks and troughs in each spectrum per experiment. A cubic smoothingspline is fit to each spectrum, and peaks and troughs are defined as achange in the first derivative of the signal. A peak is a sign change ofthe spectrum's slope from positive to negative. Conversely, troughs aredefined as a change in sign from negative to positive. The tallest peakwithin a window at the expected location of the free TNF-Alexa532 peak(e.g., 11.5 to 13 minutes) is taken to be the free peak itself. Thetroughs directly above and below the detected free peak define the upperand lower limits of the peak itself. Areas under the bound, free (TNFand IFX) and negative control peaks are found by integrating the peakarea within the limits described above using the trapezoid rule. The %of the TNF-Alexa532 peak area is then calculated for each sample byusing the formula:

%=[(a−b)/c]*100

wherein a=AUC of the TNF-Alexa532 peak in an unknown sample, b=AUC ofthe TNF-Alexa532 peak from a NAb negative control (e.g.,IFX-Alexa488+TNF-Alexa532 in normal human serum), and c=AUC of the freeTNF-Alexa532 in normal human serum. For the calculation, “c” is set to100% and “b” is as close to 0% as possible, although it may vary basedon reaction conditions. The range between “b” and “c” defines themaximum window of sensitivity.

Results:

The NAb assay of the invention has demonstrated high reproducibility,accuracy, and precision. The intra- and inter-assay precision is lessthan 20% of CV, and the accuracy of the assay is within 25%. Theprecision and accuracy obtained with the NAb assay of the invention issubstantially better than cell-based assays or ELISAs. IFX drugtolerance is ˜6 μg/mL, while TNFα interferes at greater than 1.0 ng/mL.Positive controls from pooled ATI positive patient serum dilute linearlyfrom 40-5% NAb. Analysis of healthy controls shows that samples thatreturn a value of >3% (e.g., 3.06%) are considered NAb positive. Morethan 30 ATI positive patient serum samples (3.12-199.43 U/mL) werescreened for NAb, and 26 out of 132 (19.7%) of the ATI positive patientserum samples were NAb positive (mean 22.47%, range 3.29-51.63%). ATIlevels greater than 60 U/mL corresponded to highly neutralizing Ab.Further analysis of NAb positive samples reveals a linear correlationbetween ATI titer and NAb positivity. In particular, FIG. 1 illustratesthat there was a clear relationship between NAb percent (y-axis) and ATIlevels (Spearman Rank Correlation, rho=0.564, p<<0.0001). FIG. 2illustrates that an ATI concentration ≧60 U/ml is predictive of NAbpositivity (NAb+). Sensitivity=77.8%; Specificity=98.1%; Oddsratio=63.6, p<<0.0001, Fisher's Exact Test. FIG. 3 illustrates an ATIcutoff analysis and demonstrates that ATI predicts NAb with a ROC AUC of0.931. True Positive Rate (TPR)=Sensitivity; False Positive Rate(FPR)=1−Specificity.

Conclusion:

Monitoring of NAb, in addition to drug and ADA levels, providesnecessary information on the ADA response and helps guide earlytherapeutic intervention. This method can be applied to thecharacterization of ADA against any biologic therapy.

Example 2 Patient Case Studies for Monitoring the Formation ofNeutralizing Anti-Drug Antibodies Over Time

This example illustrates additional embodiments of a novel homogeneousassay for detecting or measuring the presence or level of neutralizingand/or non-neutralizing anti-drug autoantibodies (ADA) in a patientsample (e.g., serum) using size exclusion chromatography in the presenceof labeled (e.g., fluorescently labeled) anti-TNFα drug and labeledTNFα. In addition, this example demonstrates time course case studies ofIBD patients on anti-TNFα drug therapy for monitoring the formation ofneutralizing and/or non-neutralizing anti-drug antibodies and/or a shiftfrom non-neutralizing to neutralizing anti-drug antibodies while thepatient is on therapy.

1. Drug and Anti-Drug Antibody Assays

FIG. 4 illustrates detection of ATI (i.e., antibody to IFX; “HACA”) bythe fluid phase mobility shift assay described herein. For example, 444ng of Alexa488 labeled IFX (18.8 μg/ml in 100% serum) was spiked into asample to outcompete free IFX. In particular embodiments, patient serumsamples containing complexes of IFX and ATI can be subjected to aciddissociation, wherein equilibration with acid dissociation and labeladdition followed by neutralization is performed.

FIG. 5 illustrates an exemplary ATI/IFX fluid phase mobility shift assayof the present invention. For example, samples containing variousconcentrations of ATI (standards or unknowns) equilibrated withfluorescently labeled Infliximab (IFX-488) were injected on sizeexclusion columns in 2% serum. FIG. 5 shows that large IFX-488/ATIcomplexes eluted first, followed by smaller complexes and then unboundIFX-488 and the Alexa488 loading control. Unknown concentrations weredetermined by interpolation from a standard curve. Detection of IFXfollowed a similar methodology.

2. Neutralizing and Non-Neutralizing Anti-Drug Antibody Assays

FIGS. 6 and 7 illustrate assays of the present invention for determiningwhether anti-drug antibodies such as ATI are neutralizing ornon-neutralizing autoantibodies using size exclusion chromatography todetect the binding of these autoantibodies to fluorescently labeledanti-TNFα drug in the presence of fluorescently labeled TNFα. In oneexemplary embodiment, an anti-TNFα drug such as IFX is labeled with afluorophore “F1”, wherein the fluorophore can be detected on either orboth the visible and IR spectra. Similarly, TNFα is labeled with afluorophore “F2”, wherein the fluorophore can also be detected on eitheror both the visible and IR spectra, and wherein “F1” and “F2” aredifferent fluorophores. The labeled anti-TNFα drug and the labeled TNFαare incubated with human serum in a liquid phase reaction to allow theformation of complexes (i.e., immune complexes) between the labeledanti-TNFα drug (e.g., IFX), labeled TNFα, and/or anti-drug antibodies(e.g., ATI) present in the serum.

Following incubation, the samples are loaded directly onto a sizeexclusion column and subjected to the HPLC mobility shift assay. FIG. 6illustrates a non-neutralizing anti-drug antibody (ADA) assay of thepresent invention in which binding of both the anti-drug antibody (e.g.,ATI) and the labeled TNFα (e.g., Alexa532 labeled TNFα; “TNF-532”) tothe labeled anti-TNFα drug (e.g., Alexa488 labeled IFX; “IFX-488”)results in a decrease in free TNF-532 levels. FIG. 7 illustrates aneutralizing ADA assay of the present invention in which binding ofanti-drug antibody (e.g., ATI) to the labeled anti-TNFα drug (e.g.,IFX-488) without binding of the labeled TNFα (e.g., TNF-532) results insubstantially the same amount of free TNF-532 levels as the TNF-532control.

3. Time Course Studies for Monitoring Neutralizing and Non-NeutralizingAnti-Drug Antibodies

FIGS. 8-11 illustrate data from a UC patient case study for determiningwhether anti-drug antibodies such as ATI are neutralizing ornon-neutralizing autoantibodies using the mobility shift assays of thepresent invention. For example, FIG. 8 illustrates the levels of IFX andATI over a time course of 5 samples taken 1, 2, or 3 months apart. FIG.9 shows peak analysis to determine the percentage of free TNFα overtime. In particular, the peak area of TNF-532/IFX-488 complexes wassubtracted from the free labeled TNFα area of all samples and then % offree TNFα was calculated. Notably, FIG. 9 demonstrates an increase inthe level of free TNFα over the time course of 5 samples taken 1, 2, or3 months apart, indicating an increase in neutralizing autoantibodylevels. FIG. 10 illustrates a shift from the presence ofnon-neutralizing autoantibodies to neutralizing autoantibodies over timeas exemplified in 3 samples taken 2 or 3 months apart and spiked withIFX. For the “Nov Year 1” sample, non-neutralizing antibody binds tospiked-in IFX and shows a decrease in the TNF-532 peak. For the “JanYear 2” sample, a mixture of neutralizing antibody(NAb)/non-neutralizing antibody (Ab) shows a small decrease in theTNF-532 peak relative to the level of the initial complex. As ATIbecomes almost completely neutralizing (“April Year 2” sample), high IFXlevels cannot overcome ATI binding to IFX, preventing any TNFα binding.As such, FIG. 10 demonstrates a UC patient ATI profile in which the ATIprofile shifts from a non-neutralizing ATI profile to a profilecontaining a mixture of neutralizing ATI and non-neutralizing ATI to aneutralizing ATI profile over the course of IFX therapy. FIG. 11 showspeak analysis to determine the percentage of free TNFα over time insamples that were spiked with IFX. In particular, the peak area ofTNF-532/IFX-488 complexes was subtracted from the free TNFα area of allsamples and then the percent (%) of free TNFα was calculated. Notably,FIG. 11 demonstrates an increase in the level of free TNFα over the timecourse of samples taken from the UC patient, indicating an increase inneutralizing autoantibody levels and a shift from non-neutralizing ATIto neutralizing ATI while the patient is on IFX therapy.

FIGS. 12-14 illustrate various controls performed using the mobilityshift assays of the present invention. In particular, FIG. 12 shows theuse of rabbit anti-human IgG1 Fc as a non-neutralizing antibody (Ab)control. FIG. 13 shows the use of ATI positive serum as a mixedneutralizing antibody (NAb)/non-neutralizing antibody (Ab) control. FIG.14 shows that purification of ATI from ATI positive serum results inloss of weaker affinity NAb. FIG. 15 illustrates peak analysis from a UCpatient case study to determine the percentage of free TNFα in thesevarious controls. In particular, the peak area of the TNF-532/IFX-488complex was subtracted from the free TNFα area of all samples and thenthe percent (%) of free TNFα was calculated.

FIGS. 16-18 illustrate data from CD patient case studies for determiningwhether anti-drug antibodies such as ATI are neutralizing ornon-neutralizing autoantibodies using the mobility shift assays of thepresent invention. For example, FIG. 16 shows a peak analysis from a CDpatient case study to determine the percentage of free TNFα over a timecourse of 4 samples taken 7 or 8 weeks apart during a 30-week period.Moreover, FIG. 17 shows a peak analysis from another CD patient casestudy to determine the percentage of free TNFα over a time course of 3samples taken during a 50-week period. In addition, FIG. 18 shows a peakanalysis from 4 additional CD patient case studies to determine thepercentage of free TNFα in a sample at a particular week during or afterinduction or maintenance of therapy.

Example 3 Detection of Neutralizing Antibody (NAb) Activity via an HPLCMobility Shift Competitive Ligand-Binding Assay

This example illustrates yet additional embodiments of a novelhomogeneous assay for detecting or measuring the presence or level ofneutralizing and/or non-neutralizing anti-drug autoantibodies (ADA) in apatient sample (e.g., serum) using an HPLC size exclusion chromatographyassay. In addition, this example demonstrates methods for predictingand/or determining the cross-reactivity of NAb with alternativebiological drugs such as other anti-TNF drugs.

In some embodiments, a multi-tiered approach to immunogenicity testingcomprises first screening both drug and anti-drug antibodies by a rapid,sensitive screening assay. This approach is recommended by both the FDAand the EMEA and is a useful management tool for large clinical trialsand multiple time points per patient. After confirming the presence ofADA such as ATI, patient samples are then further examined for thepresence of neutralizing antibodies that may have significant negativeclinical consequences. Neutralizing antibodies interfere with thebiological activity by binding to or near the active site, or byinduction of conformational changes, inducing a loss of efficacy.Samples containing ATI may also be screened for isotype and epitopespecificity. Comparison of patients' clinical responses to productbefore and following ADA development can provide information on thecorrelation between ADA development (and antibody characteristics) andclinical responses.

A NAb assay has been developed as disclosed herein that utilizes an HPLCmobility shift assay. In certain embodiments, the multi-tiered approachor test comprises or consists of any one, two, or all three of thefollowing tiers: (1) screening to qualitatively determine if a sample isNAb positive (yes/no based on cutpoint established from analysis ofnormal human serum); (2) confirming that the sample is NAb positiveusing, e.g., immunocompetition and/or immunodepletion; and/or (3)predicting and/or determining the cross-reactivity of NAb withalternative biological drugs.

I. Screening Tier

After a patient sample has been confirmed as positive for ADA, it can bescreened for NAb. In certain aspects, a subpopulation of ADA is NAb. Incertain embodiments, patient serum containing ADA (e.g., antibody toIFX, also known as “ATI” or “HACA”) is first acid dissociated with 0.5Mcitric acid in HPLC water for 1 hr at room temperature (RT). Samples areprepared in a 96 well plate and incubation is conducted in the dark on aplate shaker. Next, two labeled proteins (e.g., drug-Alexa488 (e.g.,IFX-Alexa488) and TNFα-Alexa532 in HPLC water containing 0.1% BSA) areadded. The samples are neutralized by the addition of 10×PBS, pH 7.3,and incubation for 1 hour at RT in the dark on a plate shaker. Thesamples are diluted to 2% serum with additional 10× buffer and HPLCwater. The samples are then injected by HPLC on a size exclusion column.Complexes or species of differing sizes are separated and monitored byfluorescence, e.g., Free TNFα-Alexa532 (“TNF532”), Free IFX-Alexa488(“IFX488”), TNF532/IFX488 complexes, TNF532/IFX488/ATI complexes(non-neutralizing Ab), and ATI/IFX488 complexes (NAb). After comparingthe results to negative (see, e.g., FIGS. 12, 19) and positive (see,e.g., FIG. 13) controls along with a cutoff established from normalhuman sera (e.g., reference range of 3.06% NAb), the sample can bedesignated as positive or negative for NAb and a titer can be determined

FIG. 19 demonstrates detection of non-neutralizing antibody activity viathe mobility shift assay. Upon combination of TNF532 with IFX488, thereis a shift to the retention time of approximately 8 minutes, indicatingthe formation of a higher molecular weight complex. The Free IFX-488peak (around 10.5 minutes) completely disappears and the Free TNF-532peak (around 12 minutes) almost completely disappears as well(indicating the formation of an ATI/IFX/TNF ternary complex). Anon-neutralizing Ab that binds away from the active site of IFX followsa similar pattern. The mouse monoclonal antibody (e.g., around 7minutes) performs as desired.

FIG. 13 demonstrates detection of neutralizing antibody activity via themobility shift assay. A completely neutralizing Ab prevents the abilityof IFX to bind to TNF (e.g., due to blockage of the active site). Thisis seen in the chromatogram as a disappearance of the IFX-488 peak withthe formation of a higher molecular weight species. The TNF-532 peakwill not change. In reality, most patients experience a combination ofnon-neutralizing/neutralizing Ab as seen in the pooled patient serum inFIG. 13 (ATI Pos. Serum, solid black line). Rabbit polyclonal antibodiesagainst the F(ab′)2 fragment of IFX/Humira as an improved NAb positivecontrol are also useful.

FIG. 8 illustrates the development of a NAb response over time in apatient during the course of IFX treatment. While they are positive forATI at all time points, it is not until the Jan Year 2 (light greyarrow, third from top at ˜12 min) time point that NAb develops. TheATI/IFX-488 complexes shift to a slightly different retention time (˜7.8minutes) that indicates a different sized complex as compared tocomplexes of TNF532/IFX488/ATI (˜8.2 and 8.8 mins). Confirmation ofneutralizing activity in the presence of additional IFX versus anirrelevant protein (immunocompetition) may be performed as well.Patients such as this would be ideal candidates for treatmentadjustment.

FIG. 9 plots the data as a bar graph of the AUC of the % free TNF peakremaining, clearly demonstrating that over time the patient isdeveloping NAb. Even low levels of NAb development observed at earlytime points are predictive of disease relapse; treatment adjustment forpatients displaying this activity is recommended. For example, thepatient should be placed on one or more immunosuppressive agents such asmethotrexate (MTX) or azathioprine (AZA) while taking the existinganti-TNF drug and/or switched to a different anti-TNF drug.

II. Confirmatory Tier

In the confirmatory tier, drug (e.g., anti-TNFα antibody) is spiked intothe sample at a variety of concentrations (e.g., 1-50 μg/mL) todetermine the neutralizing capability of the sample. In parallel,non-specific IgG is spiked in at similar levels. The samples spiked withdrug should show a dose response to the drug and an EC50 of the NAb canbe calculated. Non-specific IgG should have no effect Immunodepletioncan also be performed to rule out the effect of the matrix, ifnecessary.

FIG. 10 illustrates a shift from the presence of non-neutralizingautoantibodies to neutralizing autoantibodies over time as exemplifiedin 3 samples taken 2 or 3 months apart and spiked with IFX. Patientserum from each time point responds to spiked-in IFX, showingspecificity of response. Over time, the NAb becomes more neutralizingand eventually can neutralize >20 μg/mL IFX (the April Year 2 sampledoes not decrease when IFX is spiked-in). A complete titration can beperformed to determine the EC50 of the NAb at each time point.

III. Cross-Reactivity Tier

The cross-reactivity tier is particularly useful for predicting whethera patient will respond to a drug or therapy such as, e.g., an anti-TNFαdrug or therapy.

In some embodiments, the present invention provides methods to rapidlydetermine which therapeutic drugs will or will not work in a patient(e.g., a Crohn's disease, ulcerative colitis, or rheumatoid arthritispatient) based on the ability of an anti-drug antibody (ADA) tocross-react with a series of different anti-TNF therapeutics. As anon-limiting example, one or more of the following drugs may be testedin patients (e.g., Crohn's disease, ulcerative colitis, and/orrheumatoid arthritis patients) that have NAb to Remicade (infliximab):Enbrel (etanercept); Humira (adalimumab); Cimzia (certolizumab pegol);and Simponi (golimumab). After testing positive for NAb with a specificdrug (e.g., IFX), the NAb assay can then be performed with a series ofother drugs (e.g., fluorescently-labeled drugs) using the method of theinitial NAb test described above.

The predictive test of the present invention is useful in the managementof patient treatment by preventing the use of a drug (e.g., an anti-TNFαdrug) that will be neutralized by a patient's antibodies. Without beingbound by any particular theory, the sequence of the binding site of theneutralizing ADA has likely developed in such a way to resemble that ofTNFα (see, FIG. 20). If the NAb neutralizes any of the other anti-TNFdrugs, then those other anti-TNF drugs would likely be a pooralternative to the drug that is already being administered as thepatient will likely have an immune response. In some embodiments, acutoff established from normal human serum can be used to determine if atest sample from a patient is positive or negative. The test can be runin a rapid, cost-effective manner in an in vitro setting.

The following non-limiting case studies included Patients 1 and 2, whowere treated with Remicade (infliximab), but who subsequently lostresponse to Remicade. Patient 1 had UC and Patient 2 had CD. Themobility shift assay described herein clearly demonstrated that Patients1 and 2 lost response to Remicade as they developed anti-Remicadeantibodies (e.g., ATI). These anti-Remicade antibodies were then shownto be neutralizing antibodies (e.g., NAb).

FIG. 21 illustrates that Patients 1 and 2 developed neutralizingantibodies (NAb). These NAb compete with TNFα for the Remicade bindingsite. Importantly, these NAb might cross-react with other anti-TNFtherapeutics. If the NAb cross-react with other anti-TNF therapeutics,changing to another anti-TNF therapeutic will not help these patients.As such, the predictive assays of the present invention provideadvantages over current methods of managing patients who lose responseto Remicade, in which positive HACA (detectable antibody) is managed bychanging to another anti-TNF agent (see, e.g., Afif et al., Am. J.Gastroenterol., 105(5):1133-9 (2010)).

To determine the cross-reactivity of NAb produced in response to oneanti-TNF drug with other anti-TNF drugs, NAb which developed when thepatient was on Remicade (IFX) were tested against Humira (adalimumab).The data shown in FIG. 21 clearly demonstrated that NAb generatedagainst IFX cross-react with Humira FIG. 21 illustrates that the freeHumira peak (between 10 and 11 minutes, bottom panel of each patientstudy) is completely shifted to a higher molecular weight when thepatient serum containing NAb is added (˜12 minutes, patient study #1;˜12 minutes, patient study #2; bottom panel of each patient study).These results indicate that the NAb binds to Humira in such a way that,to an extent, the NAb prevents TNFα from accessing the antigen-bindingsite of Humira FIG. 22 depicts this schematically for both NAb andnon-NAb determinations.

In certain embodiments, the assay methods of the present inventionpredict that these patients will not respond to Humira or any otheranti-TNF therapeutics. The patient should not be treated with anti-TNFtherapy and should be switched to alternative therapy options,including, but not limited to, Actemra, Kineret, Orencia, Rituxan,and/or Arzerra for rheumatoid arthritis (RA), or Tysabri and/or steroidsfor Crohn's disease (CD).

Accordingly, the methods of the present invention are particularlyadvantageous for predicting whether a patient will respond to anti-TNFαtherapy by determining or measuring the presence and/or concentrationlevel of neutralizing antibodies (NAb) and/or non-NAb in a sample fromthe patient. In one embodiment, if the sample contains NAb to oneanti-TNFα drug, these NAb will likely cross-react and be neutralizing toother anti-TNFα drugs, such that the recommended treatment adjustmentfor the patient would be to switch to a drug with a different mechanismof action (e.g., a non-anti-TNF agent). In another embodiment, if thesample contains non-neutralizing ADA to one anti-TNFα drug, then therecommended treatment adjustment for the patient would be to switch toanother anti-TNFα drug.

Example 4 Assays for Detecting the Presence and Cross-Reactivity ofNeutralizing Anti-Drug Antibodies (NAb)

This example illustrates additional embodiments related to the assaymethods of the present invention for screening to determine if a sampleis NAb positive and predicting and/or determining the cross-reactivityof NAb with alternative biological drugs (see, e.g., Example 3). Inparticular embodiments, the assay methods described herein are usefulfor predicting whether a subject receiving a first anti-TNFα drug willrespond to alternative anti-TNFα therapy by determining whether a sampleobtained from the subject is either positive or negative for NAb. If thesample is positive for NAb, the methods comprise determining whether theNAb will cross-react with a second anti-TNFα drug and recommending thatthe subject be switched to a non-anti-TNFα drug when the NAb cross-reactwith the second anti-TNFα drug. If the sample is negative for NAb, themethods comprise recommending that the subject be switched to a secondanti-TNFα drug.

FIG. 23 shows the generation and use of an exemplary NAb standard curveof the invention. Samples containing various concentrations of rabbit(Rb) anti-IFX antibody (ATI) serum (i.e., standards or unknowns)equilibrated with fluorescently labeled TNF-532/IFX-488 were injectedonto size exclusion columns in 2% serum. Large immune complexes elutedfirst, followed by smaller complexes and then unbound IFX-488 andTNF-532. Unknown concentrations can be determined by interpolation fromthe standard curve. Rabbit serum containing different mixtures of NAband non-NAb can be combined to make controls. The NAb assay describedherein has an improved cut-off of 2.72% compared to an old cut-off of11.63% (N=50 normal samples). Table 2 provides a summary of NAb clinicalstudies by patient.

TABLE 2 NAb Clinical Summary-By Patient Study 1 Study 2 Study 3 n = 154n = 328 n = 64 Study (290 samples) (952 samples) (812 samples) 4 ATI+ 4373 58 30 (% total) (28%)  (22%) (91%) NAB+ 12 9 3 (23 samples) 5 (%ATI + tested) (28%)  (64%) (60%) (17%) High Nab activity 4 4 2 4 (20ug/mL) (% total) (2.6%)  (1.2%) (3.1%) (% NAB+) (33%)  (44%) (66%) (80%)

The cross-reactivity assay methods of the present invention areparticularly useful for predicting whether switching to anotherbiological treatment will be beneficial. After finding that a patient isNAb positive to one drug, fluorescently-labeled alternative drugs can beused in the assay. If patient serum still shows neutralizing capability,the new drug will be unlikely to succeed. Such methods are advantageousbecause they can be used to screen a panel of drugs in a cost-effectiveand timely manner to enable a suggestion or indication of the besttreatment options.

FIGS. 24 and 25 provide additional case studies to the patient studiesdescribed in Example 3 and set forth in FIG. 21. In particular, Patients3 and 4, who were treated with Remicade (infliximab, IFX), but whosubsequently lost response to IFX, were identified as being patients whowill likely not respond to Humira (adalimumab, ADL) because NAb whichdeveloped when the patient was on IFX were determined to becross-reactive with ADL.

FIG. 26 shows non-limiting examples of patient studies which demonstrateATI affinity maturation and the development of cross-reactive ATI.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, one of skill in the art will appreciate that certainchanges and modifications may be practiced within the scope of theappended claims. In addition, each reference provided herein isincorporated by reference in its entirety to the same extent as if eachreference was individually incorporated by reference.

1. A method for measuring the level or percent of a neutralizing form of an autoantibody to an anti-TNFα drug in a sample, the method comprising: (a) contacting the sample with a labeled anti-TNFα drug and a labeled TNFα to form: (i) a first labeled complex of the labeled anti-TNFα drug and the autoantibody; and/or (ii) a second labeled complex of the labeled anti-TNFα drug, the labeled TNFα, and the autoantibody; (b) subjecting the first labeled complex and/or the second labeled complex to size exclusion chromatography to separate them from free labeled TNFα, free labeled anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and labeled TNFα; (c) measuring the level of free labeled TNFα after size exclusion chromatography; and (d) comparing the level of free labeled TNFα measured in step (c) to a normalized level or percent of free labeled TNFα in a control sample, wherein the normalized level or percent of the free labeled TNFα in the control sample corresponds to the level or percent of a neutralizing form of the autoantibody.
 2. The method of claim 1, wherein the anti-TNFα drug is selected from the group consisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.
 3. The method of claim 1, wherein the autoantibody to the anti-TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.
 4. The method of claim 1, wherein step (c) comprises measuring the peak area of the free labeled TNFα after size exclusion chromatography.
 5. The method of claim 1, wherein the control sample is a reference sample containing only free labeled TNFα.
 6. The method of claim 1, wherein the level or percent of the free labeled TNFα in the control sample is normalized by subtracting the measured peak area of a third labeled complex formed between the labeled anti-TNFα drug and the labeled TNFα from the measured peak area of the free labeled TNFα.
 7. The method of claim 1, wherein the difference between the normalized level or percent of the free labeled TNFα in the control sample and the level of free labeled TNFα measured in step (c) corresponds to the level or percent of a non-neutralizing form of the autoantibody.
 8. The method of claim 1, wherein the sample is positive for the neutralizing form of the autoantibody when the sample has greater than or equal to about 3.00% of the neutralizing form of the autoantibody.
 9. The method of claim 1, wherein the sample is serum.
 10. The method of claim 1, wherein the sample is obtained from a subject receiving therapy with the anti-TNFα drug.
 11. The method of claim 1, wherein the labeled anti-TNFα drug and the labeled TNFα comprise different fluorophores or fluorescent dyes.
 12. A method for detecting the presence of a neutralizing and/or non-neutralizing form of an autoantibody to an anti-TNFα drug in a sample, the method comprising: (a) contacting the sample with a labeled anti-TNFα drug and a labeled TNFα to form: (i) a first labeled complex of the labeled anti-TNFα drug and the autoantibody; and/or (ii) a second labeled complex of the labeled anti-TNFα drug, the labeled TNFα, and the autoantibody; (b) subjecting the first labeled complex and/or the second labeled complex to size exclusion chromatography to separate them from free labeled TNFα, free labeled anti-TNFα drug, and/or a complex of labeled anti-TNFα drug and labeled TNFα; (c) measuring the level of free labeled TNFα after size exclusion chromatography; and (d) comparing the level of the free labeled TNFα measured in step (c) to the level of free labeled TNFα in a control sample, thereby detecting the presence of a neutralizing and/or non-neutralizing form of the autoantibody.
 13. The method of claim 12, wherein the anti-TNFα drug is selected from the group consisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.
 14. The method of claim 12, wherein the autoantibody to the anti-TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.
 15. The method of claim 12, wherein step (c) comprises measuring the peak area of the free labeled TNFα after size exclusion chromatography.
 16. The method of claim 12, wherein the control sample is a reference sample containing only free labeled TNFα.
 17. The method of claim 12, wherein a neutralizing form of the autoantibody is detected when the level of the free labeled TNFα measured in step (c) is the same or substantially the same as the level of the free labeled TNFα in the control sample.
 18. The method of claim 12, wherein a non-neutralizing form of the autoantibody is detected when the level of the free labeled TNFα measured in step (c) is decreased or absent compared to the level of the free labeled TNFα in the control sample.
 19. The method of claim 12, wherein the sample is serum.
 20. The method of claim 12, wherein the sample is obtained from a subject receiving therapy with the anti-TNFα drug.
 21. The method of claim 12, wherein the labeled anti-TNFα drug and the labeled TNFα comprise different fluorophores or fluorescent dyes.
 22. A method for determining whether a neutralizing form of an autoantibody to a first anti-TNFα drug is cross-reactive with a second anti-TNFα drug, the method comprising: (a) detecting or measuring the presence, level, or percent of a neutralizing form of the autoantibody in a sample according to a method of claim 1 to determine whether the sample is positive or negative for the neutralizing form of the autoantibody; and if the sample is positive for the neutralizing form of the autoantibody, then: (b) contacting the sample with a labeled second anti-TNFα drug to form a labeled complex of the labeled second anti-TNFα drug and the neutralizing form of the autoantibody; (c) subjecting the labeled complex to size exclusion chromatography to separate the labeled complex; and (d) detecting the labeled complex, thereby determining whether a neutralizing form of an autoantibody to a first anti-TNFα drug is cross-reactive with a second anti-TNFα drug.
 23. The method of claim 22, wherein the first and second anti-TNFα drugs are independently selected from the group consisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.
 24. The method of claim 22, wherein the autoantibody to the first anti-TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.
 25. The method of claim 22, wherein the presence of the labeled complex is an indication that the neutralizing autoantibody against the first anti-TNFα drug is cross-reactive with the second anti-TNFα drug.
 26. The method of claim 22, wherein the absence of the labeled complex is an indication that the neutralizing autoantibody against the first anti-TNFα drug is not cross-reactive with the second anti-TNFα drug.
 27. The method of claim 22, wherein the sample is serum.
 28. The method of claim 22, wherein the sample is obtained from a subject receiving therapy with the anti-TNFα drug.
 29. The method of claim 22, wherein the labeled second anti-TNFα drug comprises a fluorophore or fluorescent dye.
 30. A method for monitoring or optimizing therapy to an anti-TNFα drug in a subject receiving a course of therapy with the anti-TNFα drug, the method comprising: (a) detecting or measuring the presence, level, or percent of a neutralizing form of an autoantibody to the anti-TNFα drug according to a method of claim 1 at a plurality of time points over the course of therapy; (b) detecting a change in the presence, level, or percent of the neutralizing form of the autoantibody over time; and (c) determining a subsequent dose of the course of therapy for the subject or whether a different course of therapy should be administered to the subject based upon the change in the presence, level, or percent of the neutralizing form of the autoantibody over time.
 31. The method of claim 30, wherein the anti-TNFα drug is selected from the group consisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.
 32. The method of claim 30, wherein the autoantibody to the anti-TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.
 33. The method of claim 30, wherein the subsequent dose of the course of therapy is increased, decreased, or maintained based upon the change in the presence, level, or percent of the neutralizing form of the autoantibody over time.
 34. The method of claim 30, wherein the different course of therapy comprises a different anti-TNFα drug, the current course of therapy along with an immunosuppressive agent, or switching to a course of therapy that is not an anti-TNFα drug.
 35. The method of claim 34, wherein the different course of therapy is administered when the level or percent of the neutralizing form of the autoantibody increases over time.
 36. A method for optimizing therapy and/or reducing toxicity in a subject receiving a course of therapy with a first anti-TNFα drug, the method comprising: (a) determining whether a neutralizing form of an autoantibody to the first anti-TNFα drug is cross-reactive with a second anti-TNFα drug by detecting or measuring the presence, level, or percent of a neutralizing form of the autoantibody in a sample from the subject according to a method of claim 1; and (b) determining that a different course of therapy should be administered to the subject if the neutralizing form of the autoantibody is cross-reactive with the second anti-TNFα drug.
 37. The method of claim 36, wherein the first and second anti-TNFα drugs are independently selected from the group consisting of REMICADE™ (infliximab), ENBREL™ (etanercept), HUMIRA™ (adalimumab), CIMZIA® (certolizumab pegol), SIMPONI® (golimumab; CNTO 148), and combinations thereof.
 38. The method of claim 36, wherein the autoantibody to the first anti-TNFα drug is selected from the group consisting of a human anti-chimeric antibody (HACA), a human anti-humanized antibody (HAHA), a human anti-mouse antibody (HAMA), and combinations thereof.
 39. The method of claim 36, wherein the different course of therapy comprises switching to a course of therapy that is not an anti-TNFα drug.
 40. The method of claim 39, wherein the non-anti-TNFα drug is selected from the group consisting of an IL-6 receptor-inhibiting monoclonal antibody, anti-integrin molecule, JAK-2 inhibitor, tyrosine kinase inhibitor, nutrition therapy, and mixtures thereof.
 41. The method of claim 36, wherein the method further comprises determining that a subsequent dose of the current course of therapy should be increased or decreased, or that a different course of therapy should be administered to the subject, if the neutralizing form of the autoantibody is not cross-reactive with the second anti-TNFα drug. 